Microorganisms and methods for carbon-efficient biosynthesis of mek and 2-butanol

ABSTRACT

A non-naturally occurring microbial organism has at least one exogenous nucleic acid encoding a MEK pathway enzyme expressed in a sufficient amount to produce MEK. The MEK pathway includes an enzyme selected from an acetoacetyl-CoA dehydrogenase (bifunctional), an acetoacetyl-CoA aldehyde dehydrogenase, a 3-oxobutyraldehyde reductase, a 3-oxobutanol dehydratase, an MEK oxidoreductase, a 3-oxobutyraldehyde aminotransferase, a 4-aminobutan-2-one deaminase, a 2-amino-4-ketopentanoate (AKP) thiolase, an AKP aminotransferase, a 2,4-dioxopentanoate decarboxylase, an AKP deaminase, an acetylacrylate decarboxylase, an AKP decarboxylase, a glutamate dehydrogenase, a 3-oxobutyraldehyde oxidoreductase (aminating) and an AKP oxidoreductase (aminating). A 2-butanol pathway further includes an MEK reductase. A method for producing MEK or 2-butanol includes culturing these organisms under conditions and for a sufficient period of time to produce MEK or 2-butanol.

This application claims the benefit of priority of U.S. Provisional Application Ser. No. 61/185,969, filed Jun. 10, 2009, and U.S. Provisional Application Ser. No. 61/187,238, filed Jun. 15, 2009, each of which the entire contents are incorporated hereby by reference.

BACKGROUND OF THE INVENTION

This invention relates generally to the production of commodity and specialty chemicals and, more specifically to an integrated bioprocess for producing methyl ethyl ketone (MEK) and 2-butanol.

MEK is a four carbon ketone that is mainly used as a large volume solvent for coatings, adhesives, and inks, as well as a chemical intermediate. The main commercial route to MEK is the dehydrogenation of secondary butanol (s-butanol). The requisite s-butanol is accessible via sulphuric acid hydration of n-butene. In converting s-butanol to MEK, the alcohol vapour is fed into a multi-tubular reactor containing zinc or copper oxides as catalysts. The reaction is conducted between about 400-500° C. and at pressures of less than 4 bar. Liquid phase technology is also employed using Raney nickel or copper chromate at 150° C. Hydrogen is flashed off and the condensate is dehydrated by fractionation. The MEK phase separates from a water-ketone azeotrope obtained and is further purified by distillation.

Another method for MEK production involves the direct oxidation of n-butene in solution using palladium and cupric chlorides as catalysts. MEK can also be made as a by-product in butane-based acetic acid manufacture. Still another process has been developed in which MEK is available as a byproduct from phenol production. In this phenol manufacture, MEK and acetone are produced as byproducts.

Many of these processes are energy intensive requiring elevated temperatures. Catalyzed processes for the production of MEK can require expensive metals as well. Thus, there exists a need for compositions and methods that reduce the use of petroleum-based synthesis of MEK, as well as 2-butanol. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

In some embodiments the invention provides a non-naturally occurring microbial organism, comprising a microbial organism having a MEK pathway comprising at least one exogenous nucleic acid encoding a MEK pathway enzyme expressed in a sufficient amount to produce MEK. The MEK pathway includes an enzyme selected from the group consisting of an acetoacetyl-CoA dehydrogenase (bifunctional), an acetoacetyl-CoA aldehyde dehydrogenase, a 3-oxobutyraldehyde reductase, a 3-oxobutanol dehydratase, an MEK oxidoreductase, a 3-oxobutyraldehyde aminotransferase, a 4-aminobutan-2-one deaminase, a 2-amino-4-ketopentanoate (AKP) thiolase, an AKP aminotransferase, a 2,4-dioxopentanoate decarboxylase, an AKP deaminase, an acetylacrylate decarboxylase, an AKP decarboxylase, a glutamate dehydrogenase, a 3-oxobutyraldehyde oxidoreductase (aminating) and an AKP oxidoreductase (aminating).

In some embodiments the present invention provides a non-naturally occurring microbial organism, that includes a microbial organism having a 2-butanol pathway comprising at least one exogenous nucleic acid encoding a 2-butanol pathway enzyme expressed in a sufficient amount to produce 2-butanol. The 2-butanol pathway includes an enzyme selected from the group consisting of an acetoacetyl-CoA dehydrogenase (bifunctional), an acetoacetyl-CoA aldehyde dehydrogenase, a 3-oxobutyraldehyde reductase, a 3-oxobutanol dehydratase, an MEK oxidoreductase, an MEK reductase, an 3-oxobutyraldehyde aminotransferase, a 4-aminobutan-2-one deaminase, a 2-amino-4-ketopentanoate (AKP) thiolase, an AKP aminotransferase, a 2,4-dioxopentanoate decarboxylase, an AKP deaminase, an acetylacrylate decarboxylase, an AKP decarboxylase, a glutamate dehydrogenase, a 3-oxobutyraldehyde oxidoreductase (aminating) and an AKP oxidoreductase (aminating).

In some embodiments, the present invention provides a method for producing MEK that includes culturing a non-naturally occurring microbial organism having a MEK pathway. The pathway includes at least one exogenous nucleic acid encoding a MEK pathway enzyme expressed in a sufficient amount to produce MEK under conditions and for a sufficient period of time to produce MEK. The MEK pathway includes an enzyme selected from the group consisting of an acetoacetyl-CoA dehydrogenase (bifunctional), an acetoacetyl-CoA aldehyde dehydrogenase, a 3-oxobutyraldehyde reductase, a 3-oxobutanol dehydratase, an MEK oxidoreductase, a 3-oxobutyraldehyde aminotransferase, a 4-aminobutan-2-one deaminase, a 2-amino-4-ketopentanoate (AKP) thiolase, an AKP aminotransferase, a 2,4-dioxopentanoate decarboxylase, an AKP deaminase, an acetylacrylate decarboxylase, an AKP decarboxylase, a glutamate dehydrogenase, a 3-oxobutyraldehyde oxidoreductase (aminating) and an AKP oxidoreductase (aminating).

In some embodiments, the present invention provides a method for producing 2-butanol that includes culturing a non-naturally occurring microbial organism having a 2-butanol pathway. The pathway includes at least one exogenous nucleic acid encoding a 2-butanol pathway enzyme expressed in a sufficient amount to produce 2-butanol under conditions and for a sufficient period of time to produce 2-butanol. The 2-butanol pathway includes an enzyme selected from the group consisting of an acetoacetyl-CoA dehydrogenase (bifunctional), an acetoacetyl-CoA aldehyde dehydrogenase, a 3-oxobutyraldehyde reductase, a 3-oxobutanol dehydratase, an MEK oxidoreductase, an MEK reductase, an 3-oxobutyraldehyde aminotransferase, a 4-aminobutan-2-one deaminase, a 2-amino-4-ketopentanoate (AKP) thiolase, an AKP aminotransferase, a 2,4-dioxopentanoate decarboxylase, an AKP deaminase, an acetylacrylate decarboxylase, an AKP decarboxylase, a glutamate dehydrogenase, a 3-oxobutyraldehyde oxidoreductase (aminating) and an AKP oxidoreductase (aminating).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows pathways to MEK from acetoacetyl-CoA. Enzymes are: A) Acetoacetyl-CoA dehydrogenase (bifunctional), B) Acetoacetyl-CoA aldehyde dehydrogenase, C) 3-oxobutyraldehyde reductase, D) 3-Oxobutanol dehydratase, E) MEK oxidoreductase, F) 3-oxobutyraldehyde aminotransferase or 3-oxobutyraldehyde oxidoreductase (aminating), G) 4-Aminobutan-2-one deaminase, H) MEK reductase. Cofactors provided for each reaction are exemplary. For example, NADPH, reduced flavodoxins, reduced ferredoxins, or other electron donors, can substitute for NADH in the oxidation/reduction reactions. Similarly, alanine, asparate, glutamine, or other amino donors can substitute for glutamate.

FIG. 2 shows pathways to MEK from alanine. Enzymes are: A) AKP thiolase, B) AKP aminotransferase and/or AKP oxidoreductase (aminating), C) 2,4-Dioxopentanoate decarboxylase, D) 3-oxobutyraldehyde reductase, E) 3-Oxobutanol dehydratase, F) AKP deaminase, G) Acetylacrylate decarboxylase, H) AKP decarboxylase, I) 4-Aminobutan-2-one deaminase, J) MEK oxidoreductase, and K) MEK reductase. AKP is 2-amino-4-oxopentanoate, MEK is methyl ethyl ketone. Cofactors provided for each reaction are exemplary. For example, NADPH, reduced flavodoxins, reduced ferredoxins, or other electron donors, can substitute for NADH in the oxidation/reduction reactions. Similarly, alanine, asparate, glutamine, or other amino donors can substitute for glutamate.

FIG. 3 shows the enzymatic activities of A) 3-oxobutanol dehydratase, B) 2-(hydroxymethyl)glutarate dehydratase, C) fumarase, D) 3-dehydroquinate dehydratase, E) cyclohexanone hydratase and F) 2-keto-4-pentenoate dehydratase.

FIG. 4 shows decarboxylases (A) aconitate decarboxylase, (B) 4-oxalocrotonate decarboxylase, (C) cinnamate decarboxylase, and (D) acetylacrylate decarboxylase (theoretical).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed, in part to the design and production of cells and organisms having biosynthetic production capabilities for MEK and 2-butanol. The invention, in particular, relates to the design of microbial organisms capable of producing MEK and 2-butanol by introducing one or more nucleic acids encoding an MEK pathway enzyme.

In one embodiment, the invention utilizes in silico stoichiometric models of Escherichia coli metabolism that identify metabolic designs for biosynthetic production of MEK and 2-butanol. The results described herein indicate that metabolic pathways can be designed and recombinantly engineered to achieve the biosynthesis of MEK and downstream products such as 2-butanol in Escherichia coli and other cells or organisms. Biosynthetic production of MEK, for example, for the in silico designs are confirmed by construction of strains having the designed metabolic genotype. These metabolically engineered cells or organisms also can be subjected to adaptive evolution to further augment MEK biosynthesis, including under conditions approaching theoretical maximum growth.

In certain embodiments, the MEK biosynthesis characteristics of the designed strains make them genetically stable and particularly useful in continuous bioprocesses. Separate strain design strategies were identified with incorporation of different non-native or heterologous reaction capabilities into E. coli or other host organisms leading to MEK and 2-butanol producing metabolic pathways from either acetoacetyl-CoA or alanine. In silico metabolic designs were identified that resulted in the biosynthesis of MEK in E. coli from each of these metabolic pathways. MEK can be further processed biosynthetically to reduction product 2-butanol.

Strains identified via the computational component of the platform can be put into actual production by genetically engineering any of the predicted metabolic alterations which lead to the biosynthetic production of MEK, 2-butanol or other intermediate and/or downstream products. In yet a further embodiment, strains exhibiting biosynthetic production of these compounds can be further subjected to adaptive evolution to further augment product biosynthesis. The levels of product biosynthesis yield following adaptive evolution also can be predicted by the computational component of the system.

As used herein, the term “non-naturally occurring” when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes or proteins within a MEK or 2-butanol biosynthetic pathway.

A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides or, functional fragments thereof. Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to a microbial organism is intended to mean an organism that is substantially free of at least one component as the referenced microbial organism is found in nature. The term includes a microbial organism that is removed from some or all components as it is found in its natural environment. The term also includes a microbial organism that is removed from some or all components as the microbial organism is found in non-naturally occurring environments. Therefore, an isolated microbial organism is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments. Specific examples of isolated microbial organisms include partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or “microorganism” is intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical.

As used herein, the term “CoA” or “coenzyme A” is intended to mean an organic cofactor or prosthetic group (nonprotein portion of an enzyme) whose presence is required for the activity of many enzymes (the apoenzyme) to form an active enzyme system. Coenzyme A functions in certain condensing enzymes, acts in acetyl or other acyl group transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation.

As used herein, the term “substantially anaerobic” when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media. The term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen.

“Exogenous” as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term “endogenous” refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term “heterologous” refers to a molecule or activity derived from a source other than the referenced species whereas “homologous” refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.

The non-naturally occurring microbial organisms of the invention can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.

Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microorganism. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species. A specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase. A second example is the separation of mycoplasma 5′-3′ exonuclease and Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions. Paralogs can originate or derive from, for example, the same species or from a different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co-evolved from a common ancestor, that catalyze distinct reactions and have distinct functions in the same species. Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor. Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.

A nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species. Although generally, a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.

Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having MEK or 2-butanol biosynthetic capability, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or nonorthologous gene displacements are present in the referenced microorganism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes.

Orthologs, paralogs and nonorthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity. Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related. Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences.

Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and the following parameters: Match: 1; mismatch: −2; gap open: 5; gap extension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.

The present invention provides a non-naturally occurring microbial organism having an MEK pathway that includes at least one exogenous nucleic acid encoding an MEK pathway enzyme expressed in a sufficient amount to produce MEK. The MEK pathway includes an enzyme selected from an acetoacetyl-CoA dehydrogenase (bifunctional), an acetoacetyl-CoA aldehyde dehydrogenase, a 3-oxobutyraldehyde reductase, a 3-oxobutanol dehydratase, an MEK oxidoreductase, a 3-oxobutyraldehyde aminotransferase, a 4-aminobutan-2-one deaminase, a 2-amino-4-ketopentanoate (AKP) thiolase, an AKP aminotransferase, a 2,4-dioxopentanoate decarboxylase, an AKP deaminase, an acetylacrylate decarboxylase, an AKP decarboxylase, a glutamate dehydrogenase, a 3-oxobutyraldehyde oxidoreductase (aminating) and an AKP oxidoreductase (aminating).

In particular non-naturally occurring microbial organisms of the invention include a set of MEK pathway enzymes, wherein the set of enzymes defines a pathway to MEK. Within each set, one or more of the enzymes can be introduced exogenously. Exemplary sets of enzymes completing an MEK pathway include the following:

-   -   (1) acetoacetyl-CoA dehydrogenase (bifunctional), (2)         3-oxobutanol dehydratase, and (3) MEK oxidoreductase;     -   (1) acetoacetyl-CoA aldehyde dehydrogenase, (2)         3-oxobutyraldehyde reductase, (3) 3-oxobutanol dehydratase,         and (4) MEK oxidoreductase;     -   (1) acetoacetyl-CoA aldehyde dehydrogenase, (2)         3-oxobutyraldehyde aminotransferase and/or 3-oxobutyraldehyde         oxidoreductase (aminating), (3) 4-aminobutan-2-one deaminase,         and (4) MEK oxidoreductase;

(1) AKP thiolase, (2) AKP aminotransferase and/or AKP oxidoreductase (aminating), (3) 2,4-dioxopentanoate decarboxylase, (4) 3-oxobutyraldehyde reductase, (5) 3-oxobutanol dehydratase, (6) MEK oxidoreductase, and (7) glutamate dehydrogenase;

-   -   (1) AKP thiolase, (2) AKP deaminase, (3) acetylacrylate         decarboxylase, (4) MEK oxidoreductase and (5) glutamate         dehydrogenase; and     -   (1) AKP thiolase, (2) AKP decarboxylase, (3) 4-aminobutan-2-one         deaminase, (4) MEK oxidoreductase and (5) glutamate         dehydrogenase.

Any of the foregoing pathways to MEK can have the requisite enzyme functions provided by introduction of one or more exogenous nucleic acids, including two, three, four, five, six, seven, that is up to all the nucleic acids needed to complete a set where each nucleic acid encodes an MEK pathway enzyme. The non-naturally occurring microbial organisms of the invention that produce MEK can include at least one exogenous nucleic acid is that is a heterologous nucleic acid. Such non-naturally occurring microbial organisms can be cultured in a substantially anaerobic culture medium.

The present invention also provides a non-naturally occurring microbial organism having a 2-butanol pathway that includes at least one exogenous nucleic acid encoding a 2-butanol pathway enzyme expressed in a sufficient amount to produce 2-butanol. The 2-butanol pathway includes an enzyme selected from an acetoacetyl-CoA dehydrogenase (bifunctional), an acetoacetyl-CoA aldehyde dehydrogenase, a 3-oxobutyraldehyde reductase, a 3-oxobutanol dehydratase, an MEK oxidoreductase, an MEK reductase, an 3-oxobutyraldehyde aminotransferase, a 4-aminobutan-2-one deaminase, a 2-amino-4-ketopentanoate (AKP) thiolase, an AKP aminotransferase, a 2,4-dioxopentanoate decarboxylase, an AKP deaminase, an acetylacrylate decarboxylase, an AKP decarboxylase, a glutamate dehydrogenase, a 3-oxobutyraldehyde oxidoreductase (aminating) and an AKP oxidoreductase (aminating).

In particular non-naturally occurring microbial organisms of the invention include a set of 2-butanol pathway enzymes, wherein the set of enzymes defines a pathway to 2-butanol. Within each set, one or more of the enzymes can be introduced exogenously. Exemplary sets of enzymes completing a 2-butanol pathway include the following:

-   -   (1) acetoacetyl-CoA dehydrogenase (bifunctional), (2)         3-oxobutanol dehydratase, (3) MEK oxidoreductase, and (4) MEK         reductase;     -   (1) acetoacetyl-CoA aldehyde dehydrogenase, (2)         3-oxobutyraldehyde reductase, (3) 3-oxobutanol dehydratase, (4)         MEK oxidoreductase, and (5) MEK reductase;     -   (1) acetoacetyl-CoA aldehyde dehydrogenase, (2)         3-oxobutyraldehyde transaminase and/or 3-oxobutyraldehyde         oxidoreductase (aminating), (3) 4-aminobutan-2-one         deaminase, (4) MEK oxidoreductase, and (5) MEK reductase;     -   (1) AKP thiolase, (2) AKP aminotransferase and/or AKP         oxidoreductase (aminating), (3) 2,4-dioxopentanoate         decarboxylase, (4) 3-oxobutyraldehyde reductase, (5)         3-oxobutanol dehydratase, (6) MEK oxidoreductase, (7) MEK         reductase and (8) glutamate dehydrogenase;     -   (1) AKP thiolase, (2) AKP deaminase, (3) acetylacrylate         decarboxylase, (4) MEK oxidoreductase, (5) MEK reductase and (6)         glutamate dehydrogenase; and     -   (1) AKP thiolase, (2) AKP decarboxylase, (3) 4-aminobutan-2-one         deaminase, (4) MEK oxidoreductase, (5) MEK reductase and (6)         glutamate dehydrogenase.

Any of the foregoing pathways to 2-butanol can have the requisite enzyme function provided by introduction of one or more exogenous nucleic acids, including two, three, four, five, six, seven, eight that is up to all the nucleic acids needed to complete a set where each nucleic acid encodes a 2-butanol pathway enzyme. Additionally, the non-naturally occurring microbial organisms of the invention that produce 2-butanol can include at least one exogenous nucleic acid that is a heterologous nucleic acid. Such non-naturally occurring microbial organisms can be cultured in a substantially anaerobic culture medium.

In an additional embodiment, the invention provides a non-naturally occurring microbial organism having a MEK or 2-butanol pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of acetoacetyl-CoA to 3-oxobutanol, 3-oxobutanol to butenone, butenone to MEK, and MEK to 2-butanol. Alternatively, the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of acetoacetyl-CoA to 3-oxobutyraldehyde, acetoacetyl-CoA to 3-oxobutanol, 3-oxobutryaldehyde to 3-oxobutanol, 3-oxobutanol to butenone, butenone to MEK, and MEK to 2-butanol. In yet another alternative the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of acetoacetyl-CoA to 3-oxobutyraldehyde, 3-oxobutyraldehyde to 4-aminobutan-2-one, 4-aminobutan-2-one to butenone, butenone to MEK, and MEK to 2-butanol. Thus, the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of a MEK or 2-butanol pathway, such as those shown in FIG. 1.

In an additional embodiment, the invention provides a non-naturally occurring microbial organism having a MEK or 2-butanol pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of alanine to AKP, AKP to 2,4-dioxopentanoate, 2,4-dioxopentanoate to 3-oxobutyraldehyde, 3-oxobutryaldehyde to 3-oxobutanol, 3-oxobutanol to butenone, butenone to MEK, and MEK to 2-butanol. Alternatively, the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of alanine to AKP, AKP to acetylacrylate, acetylacrylate to butenone, butenone to MEK, and MEK to 2-butanol. In yet another alternative the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of alanine to AKP, AKP to 4-aminobutan-2-one, 4-aminobutan-2-one to butenone, butenone to MEK, and MEK to 2-butanol. Thus, the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of a MEK or 2-butanol pathway, such as that shown in FIG. 2.

While generally described herein as a microbial organism that contains a MEK or 2-butanol pathway, it is understood that the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a MEK or 2-butanol pathway enzyme expressed in a sufficient amount to produce an intermediate of a MEK or 2-butanol pathway. For example, as disclosed herein, a MEK or 2-butanol pathway is exemplified in FIGS. 1 and 2. Therefore, in addition to a microbial organism containing a MEK or 2-butanol pathway that produces MEK or 2-butanol, the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a MEK or 2-butanol pathway enzyme, where the microbial organism produces a MEK or 2-butanol pathway intermediate, for example, 3-oxobutyraldehyde, 3-oxobutanol, 4-aminobutan-2-one, butanone, acetylacrylate, or 2,4-dioxopentanoate.

It is understood that any of the pathways disclosed herein, as described in the Examples and exemplified in the Figures, including the pathways of FIGS. 1 and 2, can be utilized to generate a non-naturally occurring microbial organism that produces any pathway intermediate or product, as desired. As disclosed herein, such a microbial organism that produces an intermediate can be used in combination with another microbial organism expressing downstream pathway enzymes to produce a desired product. However, it is understood that a non-naturally occurring microbial organism that produces a MEK or 2-butanol pathway intermediate can be utilized to produce the intermediate as a desired product.

The invention is described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or proteins involved in the referenced reaction, reactant or product. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the reactants and products of the reaction.

This invention is directed, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze MEK production. Pathways for the production of MEK disclosed herein are based on common central metabolic precursors: (i) acetoacetyl-CoA, and (ii) alanine and acetyl-CoA. Successfully engineering these pathways includes identifying an appropriate set of enzymes with sufficient activity and specificity, cloning their corresponding genes into a production host, optimizing the expression of these genes in the production host, optimizing fermentation conditions, and assaying for product formation following fermentation.

In some embodiments, pathways to MEK and 2-butanol proceed from acetoacetyl-CoA. In other embodiments, pathways to MEK and 2-butanol utilize AKP thiolase, an enzyme which converts alanine and acetyl-CoA to 2-amino-4-oxopentanoate (AKP). All pathways are redox-balanced and capable of achieving the maximum theoretical MEK yield of 1.00 moles MEK per mole glucose utilized under anaerobic conditions. The pathways have an energetic yield of 1.75 mole ATP per mole glucose utilized at the maximum theoretical MEK yield.

MEK can be produced from acetoacetyl-CoA in several pathways with a minimum of three enzymatic steps as shown in FIG. 1. In the first pathway, acetoacetyl-CoA is reduced to 3-oxobutanol. This reduction is catalyzed by a bifunctional CoA-dependent aldehyde/alcohol oxidoreductase (Step A) or by separate enzymes encoding these functions (Steps B, C). 3-Oxobutanol is then dehydrated to butenone (Step D), which is then reduced to form MEK (Step E).

An alternate pathway for producing MEK from acetoacetyl-CoA proceeds through 4-aminobutan-2-one. In this pathway, acetoacetyl-CoA is first reduced to 3-oxobutyraldehyde by acetoacetyl-CoA aldehyde dehydrogenase, a CoA-dependent oxidoreductase (Step B). 3-Oxobutyraldehyde is then converted to 4-aminobutan-2-one by an aminotransferase or an aminating oxidoreductase (Step F). The product 4-aminobutan-2-one is deaminated to butenone (Step G), which is then reduced to form MEK (Step E).

The routes detailed in FIG. 1 are capable of achieving an MEK yield of 1.0 moles per mole glucose utilized (0.40 g/g) in E. coli. The energetic yield of the pathways is 1.75 moles ATP per mol glucose utilized at the maximum MEK yield. Product and energetic yield calculations assume: 1) pyruvate formate-lyase and/or pyruvate dehydrogenase is functional, 2) a glutamate dehydrogenase is present in the host organism, and 3) MEK exits the cell by a diffusive mechanism.

The routes described above for generating an MEK-producing organism from acetoacetyl-CoA can also be applied to produce 2-butanol, when an enzyme with MEK reductase activity is present. 2-Butanol-producing organisms are capable of achieving a product yield of 1 mole of 2-butanol per mole of glucose utilized (0.41 g/g) in E. coli. Assuming that 2-butanol exits the cell by diffusion, this pathway has favorable energetics, with an ATP yield of 2 moles ATP per mole glucose utilized at the maximum 2-butanol yield of 1 mol/mol. The assumptions used to calculate the MEK yield also apply here.

The conversion of alanine to MEK can be accomplished by a number of pathways requiring a minimum of four enzymatic steps as shown in FIG. 2. In the first step of all of these pathways alanine and acetyl-CoA are joined by 2-amino-4-ketopentanoate (AKP) thiolase, a highly selective enzyme. The product of this reaction, 2-amino-4-oxopentanoate (AKP) can then be transaminated, reduced, decarboxylated or deaminated as shown in FIG. 2.

In one route, an aminotransferase or an aminating oxidoreductase (Step B) converts AKP to 2,4-dioxopentanoate, a 2-keto acid similar in structure to alpha-ketoglutarate. 2,4-dioxopentanoate is then converted to 3-oxobutyraldehyde by a 2-ketoacid decarboxylase (Step C). 3-oxobutyraldehyde is then reduced to 3-oxobutanol (Step D), which can then be dehydrated and reduced to form MEK (Steps E and J). In an alternative route, AKP is deaminated to acetylacrylate (Step F). Acetylacrylate is decarboxylated to butenone (Step G), which is then converted to MEK by MEK oxidoreductase (Step J). Yet another route entails decarboxylation of AKP by an amino acid decarboxylase (Step H). The decarboxylation product, 4-aminobutan-2-one, is then deaminated to butenone (Step I). The deamination product, butenone, is then reduced to MEK (Step J).

The routes detailed in FIG. 2 are capable of achieving an MEK yield of 1.0 mol/mol under anaerobic conditions (0.40 g/g) in E. coli. The energetic yield of the pathways is 1.75 moles ATP per mol glucose utilized at the maximum MEK yield. Yield calculations were performed in E. coli and assume the host organism contains: 1) a functional pyruvate formate-lyase and/or pyruvate dehydrogenase, and 2) a glutamate dehydrogenase that utilizes NADH as a cofactor, 3) a pathway for making alanine, and 4) diffusive transport of the MEK product.

The three routes described above for generating an MEK-producing organism from alanine can also be applied to produce 2-butanol, when an enzyme with MEK reductase activity is present. 2-Butanol-producing organisms are capable of achieving a product yield of 1 mole of 2-butanol per mole of glucose utilized (0.41 g/g) in E. coli. Assuming that 2-butanol exits the cell by passively diffusion, this pathway has favorable energetics, with an ATP yield of 2 moles ATP per mole glucose utilized at a 2-butanol yield of 1 mol/mol. The assumptions used to calculate the MEK yield also apply here.

Enzymes that carry out the transformation shown in FIGS. 1 and 2 are described below. In some embodiments, additional genes are provided for glutamate dehydrogenase, an enzyme that can improve the MEK and/or 2-butanol yields.

In some embodiments, the non-naturally occurring microbial organism has an acetoacetyl-CoA dehydrogenase (bifunctional) encoded by a gene selected from adhE, adhE2, mcr, Rcas_(—)2929, NAP1_(—)02720, MGP2080_(—)00535, and FAR.

The conversion of acetoacetyl-CoA to 3-oxobutanol, step A of FIG. 1, is catalyzed by acetoacetyl-CoA dehydrogenase, a bifunctional CoA-dependent alcohol forming oxidoreductase. Exemplary two-step oxidoreductases that convert an acyl-CoA to an alcohol include those that transform substrates such as acetyl-CoA to ethanol (e.g., adhE from Escherichia coli (Kessler et al., FEBS Lett. 281:59-63 (1991)) and butyryl-CoA to butanol (e.g. adhE2 from Clostridium acetobutylicum (Fontaine et al., J. Bacteriol. 184:821-830 (2002)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J. Gen. Appl. Microbiol. 18:3-55 (1972); Koo et al., Biotechnol. Lett. 27:505-510 (2005)). A summary of useful GenBank information related to these genes is shown below in Table 1.

TABLE 1 adhE NP_415757.1 Escherichia coli adhE2 AAK09379.1 Clostridium acetobutylicum adhE AAV66076.1 Leuconostoc mesenteroides

Another exemplary enzyme can convert malonyl-CoA to 3-HP. An NADPH-dependent enzyme with this activity has characterized in Chloroflexus aurantiacus where it participates in the 3-hydroxypropionate cycle (Hugler et al., J. Bacteriol. 184:2404-2410 (2002); Straus and Fuchs, Eur. J. Biochem. 215:633-643 (1993)). This enzyme, with a mass of 300 kDa, is highly substrate-specific and shows little sequence similarity to other known oxidoreductases (Hugler, supra). There are indications through bioinformatics that other organisms have similar pathways (Klatt et al., Environ. Microbiol. 9:2067-2078 (2007)). Enzymes in other organisms including Roseiflexus castenholzii, Erythrobacter sp. NAP1 and marine gamma proteobacterium HTCC2080 can be inferred by sequence similarity. A summary of useful GenBank information related to these genes is shown below in Table 2.

TABLE 2 mcr AAS20429.1 Chloroflexus aurantiacus Rcas_2929 YP_001433009.1 Roseiflexus castenholzii NAP1_02720 ZP_01039179.1 Erythrobacter sp. NAP1 MGP2080_00535 ZP_01626393.1 marine gamma proteobacterium HTCC2080

Longer chain acyl-CoA molecules can be reduced by enzymes such as the jojoba (Simmondsia chinensis) FAR (GenBank AAD38039.1) which encodes an alcohol-forming fatty acyl-CoA reductase (FAR). Its overexpression in E. coli resulted in FAR activity and the accumulation of fatty alcohol (Metz et et al., Plant Physiology 122:635-644 (2000)).

In some embodiments, the non-naturally occurring microbial organism has an acetoacetyl-CoA aldehyde dehydrogenase encoded by a gene selected from acr1, sucD, bphG, adhE, Msed_(—)709, mcr, asd-2, Saci_(—)2370, Ald, and eutE.

The reduction of acetoacetyl-CoA to 3-oxobutyraldehyde can be performed by a CoA-dependent aldehyde dehydrogenase. Several acyl-CoA dehydrogenases are capable of reducing an acyl-CoA to its corresponding aldehyde. Exemplary genes that encode such enzymes include the Acinetobacter calcoaceticus acr1 encoding a fatty acyl-CoA reductase (Reiser and Somerville, J. Bacteriology 179:2969-2975 (1997)), the Acinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 (2002)), and a CoA- and NADP-dependent succinate semialdehyde dehydrogenase encoded by the sucD gene in Clostridium kluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)). SucD of P. gingivalis is another succinate semialdehyde dehydrogenase (Takahashi et al., J. Bacteriol. 182:4704-4710 (2000)). The acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yet another enzyme that can be used as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski et al., J. Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J. Gen. Appl. Microbiol. 18:43-55 (1972); Koo et al., Biotechnol. Lett. 27:505-510 (2005)). A summary of useful GenBank information related to these genes is shown below in Table 3.

TABLE 3 acr1 YP_047869.1 Acinetobacter calcoaceticus acr1 AAC45217 Acinetobacter baylyi acr1 BAB85476.1 Acinetobacter sp. Strain M-1 sucD P38947.1 Clostridium kluyveri sucD NP_904963.1 Porphyromonas gingivalis bphG BAA03892.1 Pseudomonas sp adhE AAV66076.1 Leuconostoc mesenteroides

An additional enzyme that converts an acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaeal bacteria (Berg et al., Science 318:1782-1786 (2007); Thauer, R. K., Science 318:1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus spp (Alber et al., J. Bacteriol. 188:8551-8559 (2006); Hugler et al., J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed_(—)0709 in Metallosphaera sedula (Alber et al., supra; Berg et al., Science 318:1782-1786 (2007)). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al., supra). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WO 2007/141208). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzymes have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional genes can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius and have been listed below. Yet another gene for CoA-acylating aldehyde dehydrogenase is the ald gene from Clostridium beijerinckii (Toth et al., Appl. Environ. Microbiol. 65:4973-4980 (1999)). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth et al., supra). A summary of useful GenBank information related to these genes is shown below in Table 4.

TABLE 4 Msed_0709 YP_001190808.1 Metallosphaera sedula mcr NP_378167.1 Sulfolobus tokodaii asd-2 NP_343563.1 Sulfolobus solfataricus Saci_2370 YP_256941.1 Sulfolobus acidocaldarius Ald AAT66436 Clostridium beijerinckii eutE AAA80209 Salmonella typhimurium eutE P77445 Escherichia coli

In some embodiments, the non-naturally occurring microbial organism has a 3-oxobutyraldehyde reductase encoded by a gene selected from alrA, ADH2, yqhD, bdh I, bdh II, adhA, 4hbd, adhI, P84067, mmsb, dhat, and 3hidh.

The reduction of 3-oxobutyraldehyde to 3-oxobutanol can be accomplished by enzymes with 3-oxobutyraldehyde reductase functionality. Exemplary genes encoding enzymes that catalyze the conversion of an aldehyde to alcohol (i.e., alcohol dehydrogenase or equivalently aldehyde reductase) include alrA encoding a medium-chain alcohol dehydrogenase with substrate specificity for chain lengths of C2-C14 (Tani et al., Appl. Environ. Microbiol. 66:5231-5235 (2000)), ADH2 from Saccharomyces cerevisiae (Atsumi et al., Nature 451:86-89 (2008)), yqhD from E. coli which has preference for molecules longer than C3 (Sulzenbacher et al., J. Mol. Biol. 342:489-502 (2004)), and bdh I and bdh II from C. acetobutylicum which converts butyraldehyde into butanol (Walter et al., J. Bacteriol. 174:7149-7158 (1992)). The gene product of yqhD catalyzes the reduction of acetaldehyde, malondialdehyde, propionaldehyde, butyraldehyde, and acrolein using NADPH as the cofactor (Perez et al., J. Biol. Chem. 283:7346-7353 (2008)). ADH1 from Zymomonas mobilis has been demonstrated to have activity on a number of aldehydes including formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (Kinoshita et al., Appl. Microbiol. Biotechnol. 22:249-254 (1985)). A summary of useful GenBank information related to these genes is shown below in Table 5.

TABLE 5 alrA BAB12273.1 Acinetobacter sp. Strain M-1 ADH2 NP_014032.1 Saccharomyces cerevisiae yqhD NP_417484.1 Escherichia coli bdh I NP_349892.1 Clostridium acetobutylicum bdh II NP_349891.1 Clostridium acetobutylicum adhA YP_162971.1 Zymomonas mobilis

Enzymes exhibiting 4-hydroxybutyraldehyde reductase activity (EC 1.1.1.61) also fall into this category. Such enzymes have been characterized in Ralstonia eutropha (Bravo et al., J. Forensic Sci. 49:379-387 (2004)), Clostridium kluyveri (Wolff and Kenealy, Protein Express. Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz et al., J. Biol. Chem. 278:41552-41556 (2003)). Yet another gene is the alcohol dehydrogenase adhI from Geobacillus thermoglucosidasius (Jeon et al., J. Biotechnol. 135:127-133 (2008)). A summary of useful GenBank information related to these genes is shown below in Table 6.

TABLE 6 4hbd YP_726053.1 Ralstonia eutropha H16 4hbd L21902.1 Clostridium kluyveri DSM 555 4hbd Q94B07 Arabidopsis thaliana adhI AAR91477.1 Geobacillus thermoglucosidasius M10EXG

Another exemplary enzyme is 3-hydroxyisobutyrate dehydrogenase which catalyzes the reversible oxidation of 3-hydroxyisobutyrate to methylmalonate semialdehyde. This enzyme participates in valine, leucine and isoleucine degradation and has been identified in bacteria, eukaryotes, and mammals. The enzyme encoded by P84067 from Therms thermophilus HB8 has been structurally characterized (Lokanath et al., J. Mol. Biol. 352:905-917 (2005)). The reversibility of the human 3-hydroxyisobutyrate dehydrogenase was demonstrated using isotopically-labeled substrate (Manning and Pollitt, Biochem. J. 231:481-484 (1985)). Additional genes encoding this enzyme include 3hidh in Homo sapiens (Hawes et al., Methods Enzymol. 324:218-228 (2000)) and Oryctolagus cuniculus (Chowdhury et al., Biosci. Biotechnol. Biochem. 60:2043-2047 (1996); Hawes et al., supra), mmsb in Pseudomonas aeruginosa, and dhat in Pseudomonas putida (Aberhart and Hsu, J. Chem. Soc. [Perkin 1] 6:1404-1406 (1979); Chowdhury et al., Biosci, Biotechnol. Biochem. 67:438-441 (2003); Chowdhury (1996) supra). A summary of useful GenBank information related to these genes is shown below in Table 7.

TABLE 7 P84067 P84067 Thermus thermophilus mmsb P28811.1 Pseudomonas aeruginosa dhat Q59477.1 Pseudomonas putida 3hidh P31937.2 Homo sapiens 3hidh P32185.1 Oryctolagus cuniculus

In some embodiments the non-naturally occurring microbial organism has a 3-oxobutanol dehydratase encoded by one or more genes selected from hmd, BACCAP_(—)02294, ANACOL_(—)02527, NtherDRAFT_(—)2368, dmdA, dmdB, fumA, fumB, fumC, fumH, fumI, MmcB, MmcC, aroD, aroQ, and mhpD.

In step D of FIG. 1, 3-oxobutanol is dehydrated to form butenone by a 3-oxobutanol dehydratase. Several enzymes are known to catalyze the alpha, beta elimination of water. Exemplary enzymes include 2-(hydroxymethyl)glutarate dehydratase (EC 4.2.1.-), fumarase (EC 4.2.1.2), 3-dehydroquinate dehydratase (EC 4.2.1.10), cyclohexanone hydratase (EC 4.2.1.-) and 2-keto-4-pentenoate dehydratase (EC 4.2.1.80).

2-(Hydroxymethyl)glutarate dehydratase is a [4Fe-4S]-containing enzyme that dehydrates 2-(hydroxymethyl)glutarate to 2-methylene-glutarate, studied for its role in nicontinate catabolism in Eubacterium barkeri (formerly Clostridium barkeri) (Alhapel et al., Proc. Natl. Acad. Sci. USA 103:12341-12346 (2006)). Similar enzymes with high sequence homology are found in Bacteroides capillosus, Anaerotruncus colihominis, and Natranaerobius thermophilius. These enzymes are homologous to the alpha and beta subunits of [4Fe-4S]-containing bacterial serine dehydratases (e.g., E. coli enzymes encoded by tdcG, sdhB, and sdaA). An enzyme with similar functionality in E. barkeri is dimethylmaleate hydratase, a reversible Fe²⁺-dependent and oxygen-sensitive enzyme in the aconitase family that hydrates dimethylmaeate to form (2R,3S)-2,3-dimethylmalate. This enzyme is encoded by dmdAB (Alhapel et al., Proc. Natl. Acad. Sci. USA 103:12341-12346 (2006); Kollmann-Koch and Eggerer Physiol Chem. 265:847-857 (1984)). A summary of useful GenBank information related to these genes is shown below in Table 8.

TABLE 8 hmd ABC88407.1 Eubacterium barkeri BACCAP_02294 ZP_02036683.1 Bacteroides capillosus ATCC 29799 ANACOL_02527 ZP_02443222.1 Anaerotruncus colihominis DSM 17241 NtherDRAFT_2368 ZP_02852366.1 Natranaerobius thermophilus JW/NM-WN-LF dmdA ABC88408 Eubacterium barkeri dmdB ABC88409.1 Eubacterium barkeri

Fumarate hydratase (aka fumarase) enzymes naturally catalyze the reversible hydration of fumarate to malate. Although the ability of fumarate hydratase to react with 3-oxobutanol as a substrate has not been described structural information is available for this enzyme and the enzyme has been successfully engineered to alter activity, inhibition and localization (Weaver, T., Acta Crystallogr. D Biol. Crystallogr. 61:1395-1401 (2005)). E. coli has three fumarases: FumA, FumB, and FumC that are regulated by growth conditions. FumB is oxygen sensitive and only active under anaerobic conditions. FumA is active under microanaerobic conditions, and FumC is the only active enzyme in aerobic growth (Guest et al. J. Gen. Microbiol. 131:2971-2984 (1985); Tseng et al., J. Bacteriol. 183:461-467 (2001); Woods et al., Biochim. Biophys. Acta 954:14-26 (1988)). Additional enzymes are found in Campylobacter jejuni (Smith et al., Int. J. Biochem. Cell Biol. 31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch. Biochem. Biphys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi et al., J. Biochem. 89:1923-1931 (1981)). Similar enzymes with high sequence homology include fum1 from Arabidopsis thaliana and fumC from Corynebacterium glutamicum. The MmcBC fumarase from Pelotomaculum thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., FEMS Microbiol. Lett. 270:207-213 (2007)). A summary of useful GenBank information related to these genes is shown below in Table 9.

TABLE 9 fumA P0AC33 Escherichia coli K12 fumB P14407 Escherichia coli K12 fumC P05042 Escherichia coli K12 fumC O69294 Campylobacter jejuni fumC P84127 Thermus thermophilus fumH P14408 Rattus norvegicus fum1 P93033 Arabidopsis thaliana fumC Q8NRN8 Corynebacterium glutamicum MmcB YP_001211906 Pelotomaculum thermopropionicum MmcC YP_001211907 Pelotomaculum thermopropionicum

Another enzyme capable of dehydrating a beta-hydroxy ketone is 3-dehydroquinate dehydratase, also known as dehydroquinase. This enzyme reversibly dehydrates 3-dehydroquinate to form 3-dehydro-shikimate (FIG. 3, transformation A). Two distinct types of dehydroquinase, type I and type II, catalyze identical reactions but differ in amino acid composition, structure and catalytic mechanism (Gourley et al., Nat Struct. Biol. 6:521-525 (1999); Kleanthous et al., Biochem. J. 282(pt 3):687-695 (1992)). High resolution structural data is available for the type I enzyme from Salmonella typhi (Gourley et al., supra) and for the type II enzymes from Mycobacterium tuberculosis (Gourley et al., supra) and Streptomyces coelicolor (Roszak et al., Structure 10:493-503 (2002)). Dehydroquinases have also been cloned, purified and characterized in Heliobacter pylori (Bottomley, Biochem. J 319:(Pt 2):559-565 (1996)), Salmonella typhi and Escherichia coli (Chaudhuri et al., Biochem. J275:(pt 1):1-6 (1991)). A summary of useful GenBank information related to these genes is shown below in Table 10.

TABLE 10 aroD NP_416208.1 Escherichia coli K12 sp. MG1655 aroD CAA38418.1 Salmonella enterica (Salmonella typhi) aroQ NP_626225.1 Streptomyces coelicolor aroD NP_223105.1 Heliobacter pylori aroQ P0A4Z6.2 Mycobacterium tuberculosis

2-Cyclohexenone hydratase, characterized in cell extracts of Alicycliphilus denitrificans K601 (Dangel et al., Arch. Microbiol. 152:271-279 (1989)), catalyzes the dehydration of 3-hydroxycyclohexanone to 2-cyclohexenone.

Finally, 3-oxobutyraldehyde is similar to the native substrate of 2-oxopentenoate hydratase (EC 4.2.1.80). This enzyme catalyzes the dehydration of 2-keto-4-pentenoate to 2-hydroxypent-2,4-dienoate. The E. coli and Burkholderia xenovorans 2-hydroxypentadienoate hydratase was purified and characterized (Pollard and Bugg, Eur. J. Biochem. 251: 98-106 (1998); Wang and Seah FEBS J. 272:966-974 (2005)). A summary of useful GenBank information related to these genes is shown below in Table 11.

TABLE 11 mhpD P77608 Escherichia coli K12 mhpD Q13VU0 Burkholderia xenovorans (strain LB400)

An additional enzyme is 2-methylmalate dehydratase, also called citramalate hydrolase, a reversible hydrolase that catalyzes the alpha, beta elimination of water from citramalate to form mesaconate. This enzyme has been purified and characterized in Clostridium tetanomorphum (Wang and Barker, J. Biol. Chem. 244:2516-2526 (1969)). The activity of this enzyme has also been detected in several bacteria in the genera Citrobacter and Morganella in the context of the glutamate degradation VI pathway (Kato and Asano, Arch. Microbiol. 168:457-483 (1997)).

In some embodiments, the non-naturally occurring microbial organism has an MEK oxidoreductase encoded by a gene selected from NtRed1, YML131W, AtDBR1, P2, PulR, PtPPDBR, ALH1, enr, fadH, clcE, tfdFII, and macA.

Reduction of butenone (also known as 2-oxobutene) to MEK is catalyzed by MEK oxidoreductase, an NAD(P)-dependent enone reductase. Enzymes with enone reductase activity have been identified in prokaryotes, eukaryotes and plants (Shimoda et al., Bull. Chem. Soc. Jap. 77:2269-2272 (2004); Wanner and Tressl, Eur. J. Biochem. 255:271-278 (1998)). Two enone reductases from the cytosolic fraction of Saccharomyces cerevisiae were purified and characterized, and found to accept 2-oxobutene as a substrate and form MEK (Wanner and Tressl, supra). Cell extracts of cyanobacterium Synechococcus sp. PCC7942 reduced a variety of cyclic and acyclic enone substrates, including 2-oxobutene, to their corresponding alkyl ketones (Shimoda et al., supra). Enone reductases in other organisms can also catalyze this transformation.

A recombinant NADPH-dependent enone reductase from Nicotiana tabacum, encoded by NtRed1, was functionally expressed and characterized in E. coli (Matsushima et al., Bioorg. Chem. 36:23-28 (2008)). This reductase was functional on the exocyclic enoyl ketone pulegone (Matsushima et al., supra), but was not tested on 2-oxobutene as a substrate. An enzyme in S. cerevisiae at the locus YML131W, bears 30% identity to NtRed1 (evalue=1e-26). The amino acid sequence of NtRed1 shares significant homology with 2-alkenal reductase from Arabidopsis thaliana, zeta-crystallin homolog from A. thaliana, pulegone reductase from Menthe piperita and phenylpropenal alkene reductase from Pinus taeda. These enzymes are known to catalyze the reduction of alkenes of α,β-unsaturated ketones or aldehydes. A summary of useful GenBank information related to these genes is shown below in Table 12.

TABLE 12 NtRed1 BAA89423 Nicotiana tabacum YML131W AAS56318.1 Saccharomyces cerevisiae AtDBR1 NP-197199 Arabidopsis thaliana P2 CAA89262 Arabidopsis thaliana PulR AAQ75423 Menthe piperita PtPPDBR ABG91753 Pinus taeda

2-Alkenal reductase catalyzes the reduction of α,β-unsaturated double bonds of aldehydes and ketones. A barley alkenal hydrogenase ALH1 was identified with activity for wide range of substrates including 1-octene-3-one (2-oxobutene is 1-butene-3-one) (Hambraeus and Nyberg, J. Agric. Food Chem. 53:8714-8721 (2005)). The Hordeum vulgare ALH1 cDNA was cloned expressed in E. coli (Hambraeus and Nyberg, supra). A summary of useful GenBank information related to these genes is shown below in Table 13.

TABLE 13 ALH1 AAX99161 Hordeum vulgare ALH1 ACG45753 Zea mays

Enzymes with 2-enoate reductase activity (EC 1.3.1.31) can also catalyze this conversion. 2-Enoate reductase enzymes are known to catalyze the NADH-dependent reduction of a wide variety of α,β-unsaturated carboxylic acids and aldehydes (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)). The enzyme encoded by enr in C. tyrobutyricum and C. thermoaceticum (now called Moorella thermoaceticum) catalyzes the reduction of 2-butenoate to butanoate, similar to the target transformation (Geisel and Simon, Arch. Microbiol. 135:51-57 (1983); Rohdich et al., supra; Verhaert et al., Eur. J. Biochem. 187:73-79 (1990)). In the recently published genome sequence of C. kluyveri, 9 coding sequences for enoate reductases were reported, out of which one has been characterized (Seedorf et al., Proc. Natl. Acad. Sci. USA 105:2128-2133 (2008)). The enr genes from both C. tyrobutyricum and M. thermoaceticum have been cloned and sequenced and show 59% identity to each other. The former gene is also found to have approximately 75% similarity to the characterized gene in C. kluyveri (Geisel and Simon, supra). It has been reported based on these sequence results that enr is very similar to the dienoyl CoA reductase in E. coli (fadH) (Rohdich et al., supra). The C. thermoaceticum enr gene has also been expressed in a catalytically active form in E. coli (Rohdich et al., supra). A summary of useful GenBank information related to these genes is shown below in Table 14.

TABLE 14 enr ACA54153.1 Clostridium botulinum A3 str enr CAA71086.1 Clostridium tyrobutyricum enr CAA76083.1 Clostridium kluyveri enr YP_430895.1 Moorella thermoacetica fadH NP_417552.1 Escherichia coli

3-Oxoadipate oxidoreductase (maleylacetate reductase) catalyzes the reduction of 2-maleylacetate(4-oxohex-2-enedioate) to 3-oxoadipate. The reaction is very similar to the target transformation with the addition of two terminal carboxyl groups. The enzyme activity was identified and characterized in Pseudomonas sp. strain B13 (Kaschabek and Reineke, J. Bacteriol. 177:320-325 (1995); Kaschabek and Reineke, J. Bacteriol. 175:6075-6081 (1993)), and the coding gene was cloned and sequenced (Kasberg et al., J. Bacteriol. 179:3801-3803 (1997)). Genes for 3-oxoadipate oxidoreductase include cicE gene from Pseudomonas sp. strain B13 (Kasberg et al., supra), macA gene from Rhodococcus opacus (Seibert et al., J. Bacteriol. 180:3503-3508 (1998)), the macA gene from Cupriavidus necator (also known as Ralstonia eutropha 335T) (Seibert et al., Microbiology, 150:463-472 (2004)) and the tfdFII from Ralstonia eutropha JMP134 (also known as Alcaligenes eutrophus) (Seibert et al., J. Bacteriol, 180:6745-6754 (1993)). A summary of useful GenBank information related to these genes is shown below in Table 15.

TABLE 15 clcE O30847.1 Pseudomonas sp. strain B13 macA O84992.1 Rhodococcus opacus macA AAD55886 Cupriavidus necator tfdFII AAC44727.1 Ralstonia eutropha JMP134

In some embodiments the non-naturally occurring microbial organism has a 3-oxobutyraldehyde aminotransferase encoded by a gene selected from SkyPYD4, SkUGA1, UGA1, Abat, Gta-1, gabT, and puuE.

In step F of FIG. 1, 4-aminobutan-2-one is transaminated to form 3-oxobutyraldehyde. This transformation can be catalyzed by a beta-aminotransferase or an aminotransferase with preference for terminal amines. Enzymes that can carry out this reaction include beta-alanine/alpha-ketoglutarate aminotransferase, 3-amino-2-methylpropionate transaminase, and GABA aminotransferase. A beta-alanine/alpha-ketoglutarate aminotransferase for producing 3-HP from beta-alanine via malonyl-semialdehyde has been described (Jessen et al., WO/2008/027742). The gene product of SkPYD4 in Saccharomyces kluyveri was also shown to preferentially use beta-alanine as the amino group donor (Andersen and Hansen, Gene 124:105-109 (1993)). SkUGA1 encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, UGA1 (Ramos et al., Eur. J. Biochem. 149:401-404 (1985)), whereas SkPYD4 encodes an enzyme involved in both β-alanine and GABA transamination (Andersen and Hansen, supra). A summary of useful GenBank information related to these genes is shown below in Table 16.

TABLE 16 SkyPYD4 ABF58893.1 Saccharomyces kluyveri SkUGA1 ABF58894.1 Saccharomyces kluyveri UGA1 NP_011533.1 Saccharomyces cerevisiae

3-Amino-2-methylpropionate transaminase catalyzes the transformation from methylmalonate semialdehyde to 3-amino-2-methylpropionate. The enzyme has been characterized in Rattus norvegicus and Sus scrofa and is encoded by Abat (Kakimoto et al., Biochim. Biophys. Acta 156:374-380 (1968); Tamaki et al., Methods Enzymol. 324:376-389 (2000)), where it has been shown to transaminate alternate substrates including beta-alanine, 4-aminobutanoate and 3-aminobutanoate. Enzymes in other organisms with high sequence homology to the rat 3-amino-2-methylpropionate transaminase include Gta-1 in C. elegans and gabT in Bacillus subtilis. Additionally, one of the native 4-aminobutyrate (GABA) aminotransferases in E. coli, encoded by gene gabT, has been shown to have broad substrate specificity (Liu et al., Biochemistry 43:10896-10905 (2004); Schulze et al., Appl. Environ Microbiol. 56:1-6 (1990)). The gene product of puuE catalyzes the other 4-aminobutyrate transaminase in E. coli (Kurihara et al., J. Biol. Chem. 280:4602-2608 (2005)). A summary of useful GenBank information related to these genes is shown below in Table 17.

TABLE 17 Abat P50554.3 Rattus norvegicus Abat P80147.2 Sus scrofa Gta-1 Q21217.1 Caenorhabditis elegans gabT P94427.1 Bacillus subtilis gabT P22256.1 Escherichia coli K12 puuE NP_415818.1 Escherichia coli K12

In some embodiments, the non-naturally occurring microbial organism has a 4-aminobutan-2-one deaminase encoded by a gene selected from aspA and ansB.

The deamination of 4-aminobutan-2-one is deaminated to form butenone is similar to the deamination of aspartate to fumarate by aspartase. The enzyme has been studied and several crystal structures are available. The E. coli enzyme has been shown to react with alternate substrates such as aspartatephenylmethylester, asparagine, benzyl-aspartate and malate (Ma et al., Ann N.Y. Acad Sci. 672:60-65 (1992)). Directed evolution has been implemented on this enzyme to alter substrate specificity (Asano et al., Biomol. Eng. 22:95-1001 (2005). Enzymes with aspartase functionality have also been characterized in Haemophilus influenzae (Sjostrom et al., Biochim. Biophys. Acta 1324:182-190 (1997)), Pseudomonas fluorescens (Takagi et al., J. Biochem. 96:545-552 (1984)), Bacillus subtilis (Sjostrom et al., supra) and Serratia marcescens (Takagi and Kisumi, J. Bacteriol. 161:1-6 (1985)). A summary of useful GenBank information related to these genes is shown below in Table 18.

TABLE 18 aspA NP_418562 Escherichia coli K12 subsp. MG1655 aspA P44324.1 Haemophilus influenzae aspA P07346.1 Pseudomonas fluorescens ansB P26899.1 Bacillus subtilis aspA P33109.1 Serratia marcescens

A similar reaction is catalyzed by methylaspartase (EC 4.3.1.2), an enzyme participating in the glutamate fermentation route via mesaconate (Kato et al., Arch. Microbiol. 168:457-463 (1997)).

In some embodiments the non-naturally occurring microbial organism has a 2-amino-4-ketopentanoate (AKP) thiolase encoded by one or more genes selected from ortA, ortB, Amet_(—)2368, Amet_(—)2369, Teth512_(—)1478, Teth514_(—)1479, TTE1235, and thrC.

AKP thiolase (AKPT) is a pyridoxal phosphate-dependent enzyme participating in ornithine degradation in Clostridium sticklandii (Jeng et al., Biochemistry 13:2898-2903 (1974); Kenklies et al., Microbiology 145 Pt 4):819-826 (1999)). A gene cluster encoding the alpha and beta subunits of AKPT (or-2 (ortA) and or-3 (ortB)) was recently described and the biochemical properties of the enzyme were characterized (Fonknechten et al., J. Bacteriol. 191:3162-3167 (2009)). The enzyme is capable of operating in both directions and reacts with the D-isomer of alanine. Enzyme engineering or directed evolution can enable the enzyme to function with L-alanine as a substrate providing additional pathway versatility with regards to the primary substrate. AKPT from Clostridium sticklandii has been characterized but its protein sequence has not yet been published. Enzymes with high sequence homology are found in Clostridium difficile, Alkaliphilus metalliredigenes QYF, Thermoanaerobacter sp. X514, and Thermoanaerobacter tengcongensis MB4 (Fonknechten et al., supra). A summary of useful GenBank information related to these genes is shown below in Table 19.

TABLE 19 ortA YP_001086914.1 Clostridium difficile 630 ortB YP_001086915.1 Clostridium difficile 630 Amet_2368 YP_001320181.1 Alkaliphilus metalliredigenes QYF Amet_2369 YP_001320182.1 Alkaliphilus metalliredigenes QYF Teth514_1478 YP_001663101.1 Thermoanaerobacter sp. X514 Teth514_1479) YP_001663102.1 Thermoanaerobacter sp. X514 TTE1235 NP_622858.1 Thermoanaerobacter tengcongensis MB4 thrC NP_622859.1 Thermoanaerobacter tengcongensis MB4

In some embodiments, the non-naturally occurring microbial organism has an AKP aminotransferase encoded by a gene selected from aspC, AAT2, ASP5, Got2, avtA, serC, and dat.

The conversion of 2-amino-4-oxopentanoate (AKP) to 2,4-dioxopentanoate is accomplished by an enzyme in the aminotransferase family. Selection of an appropriate aminotransferase for this transformation can be dependent on the stereochemistry of the substrate. When the substrate is in the D-configuration, a D-amino acid aminotransferase (EC 2.6.1.21) can be utilized, whereas the L-stereoisomer uses an L-aminotransferase such as aspartate aminotransferase (EC 2.6.1.1).

Aspartate aminotransferase transfers an amino group from aspartate to alpha-ketoglutarate, forming glutamate and oxaloacetate. Aspartate is similar in structure to 2-amino-4-oxopentanoate. This conversion is catalyzed by, for example, the gene products of aspC from Escherichia coli (Yagi et al., FEBS Lett. 100:81-84 (1979); Yagi et al., Methods Enzymol. 113:83-89 (1985)), AAT2 from Saccharomyces cerevisiae (Yagi et al., J. Biochem. 92:34-43 (1982)) and ASP5 from Arabidopsis thaliana (de la Torree et al., Plant J. 46:414-425 (2006); Kwok and Hanson, J. Exp. Bot. 55:595-604 (2004); Wilkie and Warren, Protein Expr. Purif. 12:381-389 (1998)). The enzyme from Rattus norvegicus has been shown to transaminate alternate substrates such as 2-aminohexanedioic acid and 2,4-diaminobutyric acid (Racasens et al., Biochemistry 19:4583-4589 (1980)). Aminotransferases that work on other amino-acid-like substrates can also catalyze this transformation. Valine aminotransferase catalyzes the conversion of valine and pyruvate to 2-ketoisovalerate and alanine. The E. coli gene, avtA, encodes one such enzyme (Whalen and Berg, J. Bacteriol. 150:739-746 (1982)). This gene product also catalyzes the amination of α-ketobutyrate to generate a-aminobutyrate (Whalen and Berg, J. Bacteriol. 158:571-574 (1984)). The gene product of the E. coli serC catalyzes two reactions, phosphoserine aminotransferase and phosphohydroxythreonine aminotransferase (Lam and Winkler, J. Bacteriol. 172:6518-6528 (1990)), and activity on non-phosphorylated substrates could not be detected (Drewke et a, FEBS Lett. 390:179-182 (1996)). A summary of useful GenBank information related to these genes is shown below in Table 20.

TABLE 20 aspC NP_415448.1 Escherichia coli AAT2 P23542.3 Saccharomyces cerevisiae ASP5 P46248.2 Arabidopsis thaliana Got2 P00507 Rattus norvegicus avtA YP_026231.1 Escherichia coli serC NP_415427.1 Escherichia coli

If the substrate is present in the D-stereoisomer, transamination can be catalyzed by D-aminotransferase (EC 2.6.1.21), also known as D-amino acid aminotransferase and D-alanine aminotransferase (DAAT). This class of enzyme is noted for its broad substrate specificity, which is species-specific. The D-aminotransferase from Bacillus species YM-1, encoded by dat, has been cloned, sequenced (Tanizawa et etl., J. Biol. Chem. 264:2450-2454 (1989)) and the crystal structure has been solved (Piesach et al., Biochemistry 37:4958-4967 (1998)). This enzyme has also been engineered to alter the substrate specificity (Gutierrez et al., Eur. J. Biochem. 267:7218-7223 (2000); Gutierrez et al., Protein Eng. 11:53-58 (1998)). Additional genes are found in Bacillus licheniformis ATCC 10716 (Taylor and Fotheringham, Biochim. Biophys. Acta 1350:38-40 (1989), Staphylococcus haemolyticus (Pucci et al., J. Bacteriol. 177:336-342 (1995)) and Bacillus subtilis (Martinez-Carrion and Jenkins, J. Biol. Chem. 240:3538-3546 (1965)). A summary of useful GenBank information related to these genes is shown below in Table 21.

TABLE 21 dat P19938 Bacillus sp. YM-1 dat P54692 Bacillus licheniformis ATCC 10716 dat P54694 Staphylococcus haemolyticus dat O07597.1 Bacillus subtilis

In some embodiments, the non-naturally occurring microbial organism has a 2,4-dioxopentanoate decarboxylase encoded by a gene selected from pdc, pdc1, mdlC, dpgB, ilvB-1, kgd, and kdcA.

In step C of FIG. 2, 2,4-dioxopentanoate is decarboxylated to form 3-oxobutyraldehyde. 2,4-dioxopentanoate is similar to the native substrates of pyruvate decarboxylase (EC 4.1.1.1) and benzoylformate decarboxylase (EC 4.1.1.7). Pyruvate decarboxylase (PDC), also termed keto-acid decarboxylase, is an enzyme used in alcoholic fermentation, catalyzing the decarboxylation of pyruvate to acetaldehyde. The enzyme from Saccharomyces cerevisiae has a broad substrate range for aliphatic 2-keto acids including 2-ketobutyrate, 2-ketovalerate, 3-hydroxypyruvate and 2-phenylpyruvate (Li and Jordan, Biochemistry 38:10004-10012 (1999)). This enzyme has been engineered for altered activity, and functionally expressed in E. coli (Killenberg-Jabs et al. Eur. J. Biochem. 268:1698-1704 (2001); Li and Jordan, supra; ter Schure et al., Appl. Environ. Microbiol. 64:1301-1307 (1998)). The PDC from Zymomonas mobilus, encoded by pdc, also has a broad substrate range and has been engineered to alter the affinity for different substrates (Siegert et al., Protein Eng. Des Sel 18:345-357 (2005)). The crystal structure of this enzyme is available (Killenberg-Jabs et al., supra). Other well-characterized PDC enzymes include those from Acetobacter pasteurians (Chandra et al., Arch. Microbiol. 176:443-451 (2001)) and Kluyveromyces lactis (Krieger et al., Eur. J. Biochem. 269:3256-3263 (2002)). A summary of useful GenBank information related to these genes is shown below in Table 22.

TABLE 22 pdc P06672.1 Zymomonas mobilus pdc1 P06169 Saccharomyces cerevisiae pdc Q8L388 Acetobacter pasteurians pdc1 Q12629 Kluyveromyces lactis

Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broad substrate range and has been engineered. The enzyme from Pseudomonas putida has been studied and crystal structures of this enzyme are available (Hasson et al., Biochemistry 37:9918-9930 (1998); Polovnikova et al., Biochemistry 42:1820-1830 (2003)). Site-directed mutagenesis of two residues in the active site of the Pseudomonas putida enzyme altered the affinity (Km) of naturally and non-naturally occurring substrates (Sigert et al., supra). The properties of this enzyme have been further modified by directed engineering (Lingen et al., Protein Eng 15:585-593 (2002); Lingen et al., Chembiochem. 4:721-726 (2003)). The enzyme from Pseudomonas aeruginosa, encoded by mdlC, has also been characterized experimentally (Barrowman et al., FEMS Microbiology Lett. 34:57-60 (1986)). Additional genes from Pseudomonas stutzeri, Pseudomonas fluorescens and other organisms can be inferred by sequence homology or identified using a growth selection system developed in Pseudomonas putida (Henning et al., Appl. Environ. Microbiol 72:7510-7517 (2006)). A summary of useful GenBank information related to these genes is shown below in Table 23.

TABLE 23 mdlC P20906.2 Pseudomonas putida mdlC Q9HUR2.1 Pseudomonas aeruginosa dpgB ABN80423.1 Pseudomonas stutzeri ilvB-1 YP_260581.1 Pseudomonas fluorescens

Another enzyme capable of decarboxylating 2-oxoacids is alpha-ketoglutarate decarboxylase (KGD). The KDC from Mycobacterium tuberculosis (Tian et al., Proc. Natl Acad. Sci USA 102:10670-10675 (2005)) has been cloned and characterized. KDC enzyme activity has been detected in several species of rhizobia including Bradyrhizobium japonicum and Mesorhizobium loti (Green et al., J. Bacteriol. 182:2838-2844 (2000)). Although the KDC-encoding gene(s) have not been isolated in these organisms, the genome sequences are available and several genes in each genome are annotated as putative KDCs. A KDC from Euglena gracilis has also been characterized (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:33-38 (1991)). The first twenty amino acids starting from the N-terminus were sequenced MTYKAPVKDVKFLLDKVFKV (Shigeoka and Nakano, supra). The gene can be identified by testing genes containing this N-terminal sequence for KDC activity. A summary of useful GenBank information related to these genes is shown below in Table 24.

TABLE 24 kgd O50463.4 Mycobacterium tuberculosis kgd NP_767092.1 Bradyrhizobium japonicum USDA110 kgd NP_105204.1 Mesorhizobium loti

Yet another enzyme for catalyzing this step is branch-chain alpha-ketoacid decarboxylase (BCKA). This class of enzyme has been shown to act on a variety of compounds varying in chain length from 3 to 6 carbons (Oku and Kaneda, J. Biol. Chem. 263:18386-18396 (1988); Smit et al., Appl Environ. Microbiol 71:303-311 (2005)). The enzyme in Lactococcus lactis (kdcA, AAS49166.1) has been characterized on a variety of branched and linear substrates including 2-oxobutanoate, 2-oxohexanoate, 2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate and isocaproate (Smit et al., supra). The enzyme has been structurally characterized (Berthold et al., Acta Crystallogr. D Biol Crystallagr. 63:1217-1224 (2007)). Sequence alignments between the Lactococcus lactis enzyme and the pyruvate decarboxylase of Zymomonas mobilus indicate that the catalytic and substrate recognition residues are nearly identical (Seigert et al., Protein Eng Des Sel 18:345-357 (2005)), so this enzyme can be used for directed engineering. Decarboxylation of alpha-ketoglutarate by a BCKA was detected in Bacillus subtilis; however, this activity was low (5%) relative to activity on other branched-chain substrates (Oku and Kaneda, supra). Additional BCKA genes can be identified by homology to the Lactococcus lactis protein sequence. Many of the high-scoring BLASTp hits to this enzyme are annotated as indolepyruvate decarboxylases (EC 4.1.1.74). Indolepyruvate decarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation of indolepyruvate to indoleacetaldehyde in plants and plant bacteria.

In some embodiments, the non-naturally occurring microbial organism has an AKP deaminase encoded by a gene selected from aspA and ansB. In step 2 of this pathway, 2-amino-4-ketopentanoate is deaminated to form acetylacrylate. A similar transformation, the deamination of aspartate to fumarate, is catalyzed by aspartase. Genes for this enzyme are described above and listed in Table 18.

In some embodiments, the non-naturally occurring microbial organism has an acetylacrylate decarboxylase encoded by a gene selected from CAD, dmpH, dmpE, xylII, xylIII, Reut_B5691, Reut_B5692, pad1, pdc, pofK, padC, and pad.

In step 3, acetylacrylate is decarboxylated to 2-oxobutene(butenone). Similar reactions are catalyzed by the enzymes aconitate decarboxylase, 4-oxalocrotonate decarboxylase and cinnamate decarboxylase as shown in FIG. 4.

Aconitate decarboxylase catalyzes the final step in itaconate biosynthesis in a strain of Candida and also in the filamentous fungus Aspergillus terreus (Bonnarme et al., J. Bacteriol. 177:3573-3578 (1995); Willke and Vorlop, App. Microbiol. Biotechnol. 56:289-295 (2001)). A cis-aconitate decarboxylase (CAD) (ATEG_(—)09971, EC 4.1.1.16) has been identified and extensively studied in Aspergillus terreus and other related fungi. Recently, the gene has been cloned and functionally characterized (Kanamasa et al., Appl. Microbiol. Biotechnol. 80:223-229 (2008)) and (PCT/NL2008/050499).

4-Oxalocronate decarboxylase has been isolated from numerous organisms and characterized. Genes encoding this enzyme include dmpH and dmpE in Pseudomonas sp. (strain 600) (Shingler et al., J. Bacteriol. 174:711-724 (1992)), xylII and xylIII from Pseudomonas putida (Kato and Asano, Arch. Microbiol. 168:457-463 (1997); Lian and Whitman, J. Am. Chem. Soc. 116:10403-10411 (1994); Stanley et al., Biochemistry 39:3514 (2000))) and Reut_B5691 and Reut_B5692 from Ralstonia eutropha JMP134 (Hughes et al., J. Bacteriol. 158:79-83 (1984)). The genes encoding the enzyme from Pseudomonas sp. (strain 600) have been cloned and expressed in E. coli (Shingler et al., supra). A summary of useful GenBank information related to these genes is shown below in Table 25.

TABLE 25 dmpH CAA43228.1 Pseudomonas sp. CF600 dmpE CAA43225.1 Pseudomonas sp. CF600 xylII YP_709328.1 Pseudomonas putida xylIII YP_709353.1 Pseudomonas putida Reut_B5691 YP_299880.1 Ralstonia eutropha JMP134 Reut_B5692 YP_299881.1 Ralstonia eutropha JMP134

An additional class of decarboxylases has been characterized that catalyze the conversion of cinnamate (phenylacrylate) and substituted cinnamate derivatives to the corresponding styrene derivatives. These enzymes are common in a variety of organisms and specific genes encoding these enzymes that have been cloned and expressed in E. coli are: pad 1 from Saccharomyces cerevisae (Clausen et al., Gene 142:107-112 (1994)), pdc from Lactobacillus plantarum (Barthelmebs et al., Appl. Environ Microbiol. 67:1063-1069 (2001); Qi et al., Metab. Eng. 9:268-276 (2007); Rodriguez et al., J. Agric. Food Chem. 56:3068-3072 (2008)), pofK (pad) from Klebsiella oxytoca (Hashimoto et al., J. Biochem. 106:76-80 (1989); Uchiyama et al., Biosci Biotechnol. Biochem. 72:116-123 (2008)), Pedicoccus pentosaceus (Barthelmebs et al., supra) and padC from Bacillus subtilis and Bacillus pumilus (Cavin et al., Appl. Environ. Microbiol. 64:1466-1471 (1998)). A ferulic acid decarboxylase from Pseudomonas fluorescens also has been purified and characterized (Huang et al., J. Bacteriol. 176:5912-5918 (1994)). Importantly, this class of enzymes have been shown to be stable and do not require either exogenous or internally bound co-factors, thus making these enzymes useful for biotransformation (Sariaslani, F. S., Annu. Rev. Microbiol. 61:51-69 (2007)). A summary of useful GenBank information related to these genes is shown below in Table 26.

TABLE 26 pad1 AB368798 Saccharomyces cerevisiae pdc U63827 Lactobacillus plantarum pofK (pad AB330293 Klebsiella oxytoca padC AF017117 Bacillus subtilis pad AJ276891 Pedicoccus pentosaceus pad AJ278683 Bacillus pumilus

In some embodiments, the non-naturally occurring microbial organism has an AKP decarboxylase encoded by a gene selected from lysA, Odc1, and panD.

In step H of Figure, 2-amino-4-ketopentanoate is decarboxylated to form 4-aminobutan-2-one. This transformation is catalyzed by an amino acid decarboxylase. Selection of an appropriate decarboxylase depends on the stereochemical configuration of 4-amino-4-oxopentanoate. When this compound is in a D-configuration, a D-amino acid decarboxylase can be utilized. One such D-amino acid decarboxylase is diaminopimelate decarboxylase (DDC, EC 4.1.1.20). This enzyme catalyzes the final step of lysine biosynthesis: decarboxylation of the D-stereocenter of meso-diaminopimelate. DDC has been studied in many organisms including E. coli (Momany et al., Acta Crystallogr. D Biol. Crystallogr. 58:549-552 (2002)), Mycobacterium tuberculosis (Andersen and Hansen et al., Gene 124:105-109 (1993); Gokulan et al., J. Biol. Chem. 278:18588-18596 (2003); Kefala et al., Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 61:782-784 (2005)), Methylophilus methylotrophus (Tsujimoto et al., J. Biotechnol. 124:327-337 (2006)), and Helicobacter pylori (Hu et al., J. Biol. Chem. 283:21284-21293 (2008)). Alternately, the ornithine decarboxylase (EC 4.1.1.17) from Homo sapiens, which has a weak activity on the D-isomer of ornithine (Fitzgerald and Flanagan, DNA 8:623-634 (1989); Qu et al., Biochem. J. 375:465-470 (2003)), can be suitable for the decarboxylation in step H. A summary of useful GenBank information related to these genes is shown below in Table 27.

TABLE 27 lysA NP_417315.1 Escherichia coli lysA AAA25361.1 Mycobacterium tuberculosis lysA BAC92756.1 Methylophilus methylotrophus lysA ABW70801.1 Helicobacter pylori Odc1 AA59967.1 Homo sapiens

When 2-amino-4-ketopentanoate exhibits L-stereochemistry, an exemplary enzyme for this transformation is aspartate decarboxylase (EC 4.1.1.11). 2-Amino-4-ketopentanoate bears structural similarity to aspartate, the native substrate of this enzyme (FIG. 2). Aspartate decarboxylase participates in pantothenate biosynthesis and is encoded by gene panD in Escherichia coli (Dusch et al., Appl. Environ. Microbiol. 65:1530-1539 (1999); Merkel and Nichols, FEMS Microbiol. Lett. 143:247-252 (1996); Ramjee et al., Biochem. J. 323(Pt 3):661-669 (1997); Schmitzberger et al., EMBO J. 22:6193-6204 (2003)). The enzymes from Mycobacterium tuberculosis (Chopra et al., Pan D. Protein Expr. Purif. 25:533-540 (2002)) and Corynebacterium glutanicum (Dusch et al., supra) have been expressed and characterized in E. coli. A summary of useful GenBank information related to these genes is shown below in Table 28.

TABLE 28 panD P0A790 Escherichia coli K12 panD Q9X4N0 Corynebacterium glutanicum panD P65660.1 Mycobacterium tuberculosis

In some embodiments, thenon-naturally occurring microbial organism has an MEK reductase is encoded by a gene selected from the group consisting of sadh, adhA, and Adh.

Methyl ethyl ketone reductase, or alternatively, 2-butanol dehydrogenase, catalyzes the reduction of MEK to form 2-butanol. Exemplary enzymes can be found in Rhodococcus ruber (Kosjek et al., Biotechnol. Bioeng. 86:55-62 (2004)) and Pyrococcus furiosus (van der Oost et al., Eur. J. Biochem. 268:3062-3068 (2001)). Additional secondary alcohol dehydrogenase enzymes capable of this transformation include adh from C. beijerinckii (Hanai et al., Appl. Environ. Microbiol. 73:7814-7818 (2007); Jojima et al., Appl. Environ. Microbiol. 77:1219-1224 (2008)) and adh from Thermoanaerobacter brockii (Hanai et al., supra; Peretz et al., Anaerobe 3:259-270 (1997)). A summary of useful GenBank information related to these genes is shown below in Table 29.

TABLE 29 sadh CAD36475 Rhodococcus ruber adhA AAC25556 Pyrococcus furiosus Adh P14941.1 Thermoanaerobobacter brockii Adh AAA23199.2 Clostridium beijerinckii

In some embodiments, the non-naturally occurring microbial organism has a glutamate dehydrogenase encoded by a gene selected from GDH2, rocG, gdh1, gdh2, and GDH.

An additional gene for catalyzing the alanine pathways of FIG. 2 is an NADH-dependent glutamate dehydrogenase. This enzyme is in the EC class 1.4.1.2, and is not naturally found in E. coli. Overexpression of the NADH-dependent glutamate dehydrogenase was found to improve ethanol production in engineered strains of S. cerevisiae (Roca et al., Appl. Environ. Microbiol. 69:4732-4736 (2003)). Exemplary genes are found in Bacillus subtilis (Khan et al., Biosci. Biotechnol. Biochem. 69:1861-1870 (2005)), Nicotiana tabacum (Purnell et al., Planta 222:167-180 (2005), Oryza sativa (Abiko et al., Plant Cell Physiol. 46:1724-1734 (2005)), Haloferax mediterranei (Diaz et al., Extremophiles 10:105-115 (2006)) and Halobactreium salinarum (Hayden et al., FEMS Microbiol. Lett. 211:37-41 (2002)). The Nicotiana tabacum enzyme is composed of alpha and beta subunits encoded by gdh1 and gdh2 (Purnell et al., supra). A summary of useful GenBank information related to these genes is shown below in Table 30.

TABLE 30 GDH2 NP_010066.1 Saccharomyces cerevisiae rocG NP_391659.1 Bacillus subtilis gdh1 AAR11534.1 Nicotiana tabacum gdh2 AAR11535.1 Nicotiana tabacum GDH Q852M0 Oryza sativa GDH Q977U6 Haloferax mediterranei GDH P29051 Halobactreium salinarum

In some embodiments, the non-naturally occurring microbial organism has a 3-oxobutyraldehyde oxidoreductase (aminating) encoded by a gene selected from gdhA, gdh, gdhA1, ldh, nadX, and lysDH.

The conversion of 3-oxobutyraldehyde to 4-aminobutan-2-one can be catalyzed by an aminating oxidoreductase. Most enzymes in this class (EC 1.4.1) catalyze the oxidative deamination of alpha-amino acids with NAD+ or NADP+ as acceptor, though the reactions are typically reversible. Exemplary oxidoreductases operating on amino acids include glutamate dehydrogenase (deaminating), encoded by gdhA, leucine dehydrogenase (deaminating), encoded by ldh, and aspartate dehydrogenase (deaminating), encoded by nadX. The gdhA gene product from Escherichia coli (Korber et al., J. Mol. Biol. 234:1270-1273 (1993); McPherson and Wootton, Nucl. Acids Res. 11:5257-5266 (1983)), gdh from Thermotoga maritima (Kort et al., Extremophiles 1:52-60 (1997); Lebbink et al., J. Mol. Biol. 280:287-296 (1998); Lebbink et al., J. Mol. Biol. 289:357-269 (1999)), and gdhA1 from Halobacterium salinarum (Ingoldsby et al., Gene 349:237-244 (2005)) catalyze the reversible interconversion of glutamate to 2-oxoglutarate and ammonia, while favoring NADP(H), NAD(H), or both, respectively. The ldh gene of Bacillus cereus encodes the LeuDH protein that has a wide of range of substrates including leucine, isoleucine, valine, and 2-aminobutanoate (Ansorge and Kula, Biotechnol. Bioeng. 68:557-562 (2000); Stoyan et al., J. Biotechnol. 54:77-80 (1997)). The nadX gene from Thermotoga maritime encoding for the aspartate dehydrogenase is involved in the biosynthesis of NAD (Yang et al., J. Biol. Chem. 278:8804-8808 (2003)). A summary of useful GenBank information related to these genes is shown below in Table 31.

TABLE 31 gdhA P00370 Escherichia coli gdh P96110.4 Thermotoga maritima gdhA1 NP_279651.1 Halobacterium salinarum ldh P0A393 Bacillus cereus nadX NP_229443.1 Thermotoga maritima

The lysine 6-dehydrogenase (deaminating), encoded by the lysDH genes, catalyze the oxidative deamination of the ε-amino group of L-lysine to form 2-aminoadipate-6-semialdehyde, which in turn nonenzymatically cyclizes to form Δ¹-piperideine-6-carboxylate (Misono and Nagasaki, J. Bacteriol. 150:398-401 (1982)). Exemplary enzymes can be found in Geobacillus stearothermophilus (Heydari et al., Appl. Environ. Microbiol. 70:937-942 (2004)), Agrobacterium tumefaciens (Hashimoto et al., J. Biochem. 106:76-80 (1989); Misono and Nagasaki, supra), and Achromobacter denitrificans (Ruldeekulthamrong et al., BMB Rep 41:790-795 (2008)). Such enzymes are particularly good for converting adipate semialdehyde to 6-aminocaproate given the structural similarity between adipate semialdehyde and 2-aminoadipate-6-semialdehyde. A summary of useful GenBank information related to these genes is shown below in Table 32.

TABLE 32 lysDH BAB39707 Geobacillus stearothermophilus lysDH NP_353966 Agrobacterium tumefaciens lysDH AAZ94428 Achromobacter denitrificans

In some embodiments, the non-naturally occurring microbial organism has an AKP oxidoreductase (aminating) encoded by a gene selected from gdhA, gdh, gdhA1, ldh, nadX, and lysDH. An aminating oxidoreductase can convert AKP to 2,4-dioxopentanoate. Genes for 3-oxobutyraldehyde oxidoreducase (aminating) described above and in Tables 31 and 32, are also applicable here.

The non-naturally occurring microbial organisms of the invention can be produced by introducing expressible nucleic acids encoding one or more of the enzymes or proteins participating in one or more MEK or 2-butanol biosynthetic pathways. Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular MEK or 2-butanol biosynthetic pathway can be expressed. For example, if a chosen host is deficient in one or more enzymes or proteins for a desired biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression. Alternatively, if the chosen host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is needed for the deficient enzyme(s) or protein(s) to achieve MEK or 2-butanol biosynthesis. Thus, a non-naturally occurring microbial organism of the invention can be produced by introducing exogenous enzyme or protein activities to obtain a desired biosynthetic pathway or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produces a desired product such as MEK or 2-butanol.

Depending on the MEK or 2-butanol biosynthetic pathway constituents of a selected host microbial organism, the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed MEK or 2-butanol pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more MEK or 2-butanol biosynthetic pathways. For example, MEK or 2-butanol biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes or proteins of a MEK or 2-butanol pathway, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins. For example, exogenous expression of all enzymes or proteins in a pathway for production of MEK or 2-butanol can be included.

Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form will, at least, parallel the MEK or 2-butanol pathway deficiencies of the selected host microbial organism. Therefore, a non-naturally occurring microbial organism of the invention can have one, two, three, four, five, six, seven, eight, up to all nucleic acids encoding the enzymes or proteins constituting a MEK or 2-butanol biosynthetic pathway disclosed herein. In some embodiments, the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize MEK or 2-butanol biosynthesis or that confer other useful functions onto the host microbial organism. One such other functionality can include, for example, augmentation of the synthesis of one or more of the MEK or 2-butanol pathway precursors such as acetoacetyl-CoA or alanine.

Generally, a host microbial organism is selected such that it produces the precursor of a MEK or 2-butanol pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host microbial organism. For example, acetoacetyl-CoA is produced naturally in a host organism such as E. coli. A host organism can be engineered to increase production of a precursor, as disclosed herein. In addition, a microbial organism that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of a MEK or 2-butanol pathway.

In some embodiments, a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize MEK or 2-butanol. In this specific embodiment it can be useful to increase the synthesis or accumulation of a MEK or 2-butanol pathway product to, for example, drive MEK or 2-butanol pathway reactions toward MEK or 2-butanol production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described MEK or 2-butanol pathway enzymes or proteins. Over expression the enzyme or enzymes and/or protein or proteins of the MEK or 2-butanol pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes. Therefore, naturally occurring organisms can be readily generated to be non-naturally occurring microbial organisms of the invention, for example, producing MEK or 2-butanol, through overexpression of one, two, three, four, five, six, seven, eight, that is, up to all nucleic acids encoding MEK or 2-butanol biosynthetic pathway enzymes or proteins. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the MEK or 2-butanol biosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene's promoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non-naturally occurring microbial organism of the invention. The nucleic acids can be introduced so as to confer, for example, a MEK or 2-butanol biosynthetic pathway onto the microbial organism. Alternatively, encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer MEK or 2-butanol biosynthetic capability. For example, a non-naturally occurring microbial organism having a MEK or 2-butanol biosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins, such as the combination of an acetoacetyl-CoA dehydrogenase and a 3-oxobutanol dehydratase or an acetoacetyl-CoA aldehyde dehydrogenase and a 3-oxbutryaldehyde amino transferase, and the like. Thus, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention. Similarly, it is understood that any combination of three or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention, for example, an acetoacetyl-CoA dehydrogenase, a 3-oxobutanol dehydratase, and an MEK oxidoreductase, and so forth, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product. Similarly, any combination of four, five, six, seven, eight, or more enzymes or proteins of a biosynthetic pathway as disclosed herein can be included in a non-naturally occurring microbial organism of the invention, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product.

In addition to the biosynthesis of MEK or 2-butanol as described herein, the non-naturally occurring microbial organisms and methods of the invention also can be utilized in various combinations with each other and with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes. For example, one alternative to produce MEK or 2-butanol other than use of the MEK or 2-butanol producers is through addition of another microbial organism capable of converting a MEK or 2-butanol pathway intermediate to MEK or 2-butanol. One such procedure includes, for example, the fermentation of a microbial organism that produces a MEK or 2-butanol pathway intermediate. The MEK or 2-butanol pathway intermediate can then be used as a substrate for a second microbial organism that converts the MEK or 2-butanol pathway intermediate to MEK or 2-butanol. The MEK or 2-butanol pathway intermediate can be added directly to another culture of the second organism or the original culture of the MEK or 2-butanol pathway intermediate producers can be depleted of these microbial organisms by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps.

In other embodiments, the non-naturally occurring microbial organisms and methods of the invention can be assembled in a wide variety of subpathways to achieve biosynthesis of, for example, MEK or 2-butanol. In these embodiments, biosynthetic pathways for a desired product of the invention can be segregated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product. In such a biosynthetic scheme, the product of one microbial organism is the substrate for a second microbial organism until the final product is synthesized. For example, the biosynthesis of MEK or 2-butanol can be accomplished by constructing a microbial organism that contains biosynthetic pathways for conversion of one pathway intermediate to another pathway intermediate or the product. Alternatively, MEK or 2-butanol also can be biosynthetically produced from microbial organisms through co-culture or co-fermentation using two organisms in the same vessel, where the first microbial organism produces an MEK or 2-butanol intermediate and the second microbial organism converts the intermediate to MEK or 2-butanol.

Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the non-naturally occurring microbial organisms and methods of the invention together with other microbial organisms, with the co-culture of other non-naturally occurring microbial organisms having subpathways and with combinations of other chemical and/or biochemical procedures well known in the art to produce MEK or 2-butanol.

Sources of encoding nucleic acids for a MEK or 2-butanol pathway enzyme or protein can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction. Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. Exemplary species for such sources include, for example, Escherichia coli, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes. However, with the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the requisite MEK or 2-butanol biosynthetic activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations enabling biosynthesis of MEK or 2-butanol described herein with reference to a particular organism such as E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.

In some instances, such as when an alternative MEK or 2-butanol biosynthetic pathway exists in an unrelated species, MEK or 2-butanol biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the teachings and methods of the invention can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will synthesize MEK or 2-butanol.

Host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, yeast, fungus or any of a variety of other microorganisms applicable to fermentation processes. Exemplary bacteria include species selected from Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida. Exemplary yeasts or fungi include species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger and Pichia pastoris. E. coli is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering. Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae.

Methods for constructing and testing the expression levels of a non-naturally occurring MEK or 2-butanol-producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for production of MEK or 2-butanol can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. For exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.

An expression vector or vectors can be constructed to include one or more MEK or 2-butanol biosynthetic pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors.

For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.

The invention further provides a method for producing MEK that includes culturing a non-naturally occurring microbial organism having a MEK pathway. The pathway includes at least one exogenous nucleic acid encoding a MEK pathway enzyme expressed in a sufficient amount to produce MEK under conditions and for a sufficient period of time to produce MEK. The MEK pathway includes an enzyme selected from an acetoacetyl-CoA dehydrogenase (bifunctional), an acetoacetyl-CoA aldehyde dehydrogenase, a 3-oxobutyraldehyde reductase, a 3-oxobutanol dehydratase, an MEK oxidoreductase, a 3-oxobutyraldehyde aminotransferase, a 4-aminobutan-2-one deaminase, a 2-amino-4-ketopentanoate (AKP) thiolase, an AKP aminotransferase, a 2,4-dioxopentanoate decarboxylase, an AKP deaminase, an acetylacrylate decarboxylase, an AKP decarboxylase, a glutamate dehydrogenase, a 3-oxobutyraldehyde oxidoreductase (aminating) and an AKP oxidoreductase (aminating).

The MEK pathways include a set of MEK pathway enzymes selected from the following:

-   (1) acetoacetyl-CoA dehydrogenase (bifunctional), (2) 3-oxobutanol     dehydratase, and (3) MEK oxidoreductase; -   (1) acetoacetyl-CoA aldehyde dehydrogenase, (2) 3-oxobutyraldehyde     reductase, (3) 3-oxobutanol dehydratase, and (4) MEK oxidoreductase; -   (1) acetoacetyl-CoA aldehyde dehydrogenase, (2) 3-oxobutyraldehyde     aminotransferase and/or 3-oxobutyraldehyde oxidoreductase     (aminating), (3) 4-aminobutan-2-one deaminase, and (4) MEK     oxidoreductase; -   (1) AKP thiolase, (2) AKP aminotransferase and/or AKP oxidoreductase     (aminating), (3) 2,4-dioxopentanoate decarboxylase, (4)     3-oxobutyraldehyde reductase, (5) 3-oxobutanol dehydratase, (6) MEK     oxidoreductase, and (7) glutamate dehydrogenase ; -   (1) AKP thiolase, (2) AKP deaminase, (3) acetylacrylate     decarboxylase, (4) MEK oxidoreductase and (5) glutamate     dehydrogenase ; -   (1) AKP thiolase, (2) AKP decarboxylase, (3) 4-aminobutan-2-one     deaminase, (4) MEK oxidoreductase and (5) glutamate dehydrogenase ;

Similarly, for the production of 2-butanol, inclusion of an MEK reductase provides the following 2-butanol enzyme sets:

-   (1) acetoacetyl-CoA dehydrogenase (bifunctional), (2) 3-oxobutanol     dehydratase, (3) MEK oxidoreductase, and (4) MEK reductase; -   (1) acetoacetyl-CoA aldehyde dehydrogenase, (2) 3-oxobutyraldehyde     reductase, (3) 3-oxobutanol dehydratase, (4) MEK oxidoreductase,     and (5) MEK reductase; -   (1) acetoacetyl-CoA aldehyde dehydrogenase, (2) 3-oxobutyraldehyde     transaminase and/or 3-oxobutyraldehyde oxidoreductase     (aminating), (3) 4-aminobutan-2-one deaminase, (4) MEK     oxidoreductase, and (5) MEK reductase; -   (1) AKP thiolase, (2) AKP aminotransferase and/or AKP oxidoreductase     (aminating), (3) 2,4-dioxopentanoate decarboxylase, (4)     3-oxobutyraldehyde reductase, (5) 3-oxobutanol dehydratase, (6) MEK     oxidoreductase, (7) MEK reductase and (8) glutamate dehydrogenase; -   (1) AKP thiolase, (2) AKP deaminase, (3) acetylacrylate     decarboxylase, (4) MEK oxidoreductase, (5) MEK reductase and (6)     glutamate dehydrogenase ; and -   (1) AKP thiolase, (2) AKP decarboxylase, (3) 4-aminobutan-2-one     deaminase, (4) MEK oxidoreductase, (5) MEK reductase and (6)     glutamate dehydrogenase.

Suitable purification and/or assays to test for the production of MEK or 2-butanol can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art. For example, to assay MEK oxidoreductase activity, butenone and NADH are added to cell extracts. MEK formation is accompanied by a decrease of NADH, which is assayed by fluorescence since NADH absorbs light at a wavelength of 340 nm and radiates secondary (fluorescence) photons with a wavelength of 450 nm.

The MEK or 2-butanol can be separated from other components in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the biosynthetic products of the invention. For example, the MEK or 2-butanol producers can be cultured for the biosynthetic production of MEK or 2-butanol.

For the production of MEK or 2-butanol, the recombinant strains are cultured in a medium with carbon source and other essential nutrients. It is highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in U.S. patent application Ser. No. 11/891,602, filed Aug. 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein.

If desired, the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH. The growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.

The growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms of the invention for the production of MEK or 2-butanol.

In addition to renewable feedstocks such as those exemplified above, the MEK or 2-butanol microbial organisms of the invention also can be modified for growth on syngas as its source of carbon. In this specific embodiment, one or more proteins or enzymes are expressed in the MEK or 2-butanol producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues. Syngas is a mixture primarily of H₂ and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high fuel to oxygen ratio. Although largely H₂ and CO, syngas can also include CO₂ and other gases in smaller quantities. Thus, synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, CO₂.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ to acetyl-CoA and other products such as acetate. Organisms capable of utilizing CO and syngas also generally have the capability of utilizing CO₂ and CO₂/H₂ mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway. H₂-dependent conversion of CO₂ to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved. Many acetogens have been shown to grow in the presence of CO₂ and produce compounds such as acetate as long as hydrogen is present to supply the necessary reducing equivalents (see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation:

2CO₂+4H₂+nADP+nPi→CH₃COOH+2H₂O+nATP

Hence, non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for the production of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch. The methyl branch converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in the methyl branch are catalyzed in order by the following enzymes or proteins: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclodehydratase, methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase. The reactions in the carbonyl branch are catalyzed in order by the following enzymes or proteins: methyltetrahydrofolate:corrinoid protein methyltransferase (for example, AcsE), corrinoid iron-sulfur protein, nickel-protein assembly protein (for example, AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase and nickel-protein assembly protein (for example, CooC). Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a MEK or 2-butanol pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the Wood-Ljungdahl enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains the complete Wood-Ljungdahl pathway will confer syngas utilization ability.

Additionally, the reductive (reverse) tricarboxylic acid cycle coupled with carbon monoxide dehydrogenase and/or hydrogenase activities can also be used for the conversion of CO, CO₂ and/or H₂ to acetyl-CoA and other products such as acetate. Organisms capable of fixing carbon via the reductive TCA pathway can utilize one or more of the following enzymes: ATP citrate-lyase, citrate lyase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxide dehydrogenase, and hydrogenase. Specifically, the reducing equivalents extracted from CO and/or H₂ by carbon monoxide dehydrogenase and hydrogenase are utilized to fix CO₂ via the reductive TCA cycle into acetyl-CoA or acetate. Acetate can be converted to acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate kinase/phosphotransacetylase, and acetyl-CoA synthetase. Acetyl-CoA can be converted to the MEK/2-butanol precursors, glyceraldehyde-3-phosphate, phosphoenolpyruvate, and pyruvate, by pyruvate:ferredoxin oxidoreductase and the enzymes of gluconeogenesis. Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a MEK/2-butanol pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the reductive TCA pathway enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains the complete reductive TCA pathway will confer syngas utilization ability.

Accordingly, given the teachings and guidance provided herein, those skilled in the art will understand that a non-naturally occurring microbial organism can be produced that secretes the biosynthesized compounds of the invention when grown on a carbon source such as a carbohydrate. Such compounds include, for example, MEK or 2-butanol and any of the intermediate metabolites in the MEK or 2-butanolpathway. All that is required is to engineer in one or more of the required enzyme or protein activities to achieve biosynthesis of the desired compound or intermediate including, for example, inclusion of some or all of the MEK or 2-butanol biosynthetic pathways. Accordingly, the invention provides a non-naturally occurring microbial organism that produces and/or secretes MEK or 2-butanol when grown on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the MEK or 2-butanol pathway when grown on a carbohydrate or other carbon source. The MEK or 2-butanol producing microbial organisms of the invention can initiate synthesis from an intermediate, for example, 3-oxobutyraldehyde or AKP.

The non-naturally occurring microbial organisms of the invention are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding a MEK or 2-butanol pathway enzyme or protein in sufficient amounts to produce MEK or 2-butanol. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce MEK or 2-butanol. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of MEK or 2-butanol resulting in intracellular concentrations between about 0.1-2000 mM or more. Generally, the intracellular concentration of MEK or 2-butanol is between about 3-2000 mM, in some embodiments between about 50-1750 mM and in other embodiments between about 500-1500 mM, including about 600 mM, 900 mM, 1200 mM, 1500 mM, or more. Intracellular concentrations between and above each of these exemplary ranges also can be achieved from the non-naturally occurring microbial organisms of the invention.

In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been described previously and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S. patent application Ser. No. 11/891,602, filed Aug. 10, 2007. Any of these conditions can be employed with the non-naturally occurring microbial organisms as well as other anaerobic conditions well known in the art. Under such anaerobic conditions, the MEK or 2-butanol producers can synthesize MEK or 2-butanol at intracellular concentrations of 5-10 mM or more as well as all other concentrations exemplified herein. It is understood that, even though the above description refers to intracellular concentrations, MEK or 2-butanol producing microbial organisms can produce MEK or 2-butanol intracellularly and/or secrete the product into the culture medium.

In addition to the culturing and fermentation conditions disclosed herein, growth condition for achieving biosynthesis of MEK or 2-butanol can include the addition of an osmoprotectant to the culturing conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented as described herein in the presence of an osmoprotectant. Briefly, an osmoprotectant refers to a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress. Osmoprotectants include, but are not limited to, betaines, amino acids, and the sugar trehalose. Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate, 3-dimethylsulfonio-2-methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and ectoine. In one aspect, the osmoprotectant is glycine betaine. It is understood to one of ordinary skill in the art that the amount and type of osmoprotectant suitable for protecting a microbial organism described herein from osmotic stress will depend on the microbial organism used. The amount of osmoprotectant in the culturing conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about 10mM, no more than about 50mM, no more than about 100mM or no more than about 500mM.

The culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achieving biosynthesis of MEK or 2-butanol includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. Briefly, anaerobic conditions refers to an environment devoid of oxygen. Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation. Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an N₂/CO₂ mixture or other suitable non-oxygen gas or gases.

The culture conditions described herein can be scaled up and grown continuously for manufacturing of MEK or 2-butanol. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of MEK or 2-butanol. Generally, and as with non-continuous culture procedures, the continuous and/or near-continuous production of MEK or 2-butanol will include culturing a non-naturally occurring MEK or 2-butanol producing organism of the invention in sufficient nutrients and medium to sustain and/or nearly sustain growth in an exponential phase. Continuous culture under such conditions can include, for example, 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, organisms of the invention can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and/or near-continuous culture conditions also can include all time intervals in between these exemplary periods. It is further understood that the time of culturing the microbial organism of the invention is for a sufficient period of time to produce a sufficient amount of product for a desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of MEK or 2-butanol can be utilized in, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.

In addition to the above fermentation procedures using the MEK or 2-butanol producers of the invention for continuous production of substantial quantities of MEK or 2-butanol, the MEK or 2-butanol producers also can be, for example, simultaneously subjected to chemical synthesis procedures to convert the product to other compounds or the product can be separated from the fermentation culture and sequentially subjected to chemical conversion to convert the product to other compounds, if desired.

Directed evolution is a powerful approach that involves the introduction of mutations targeted to a specific gene in order to improve and/or alter the properties of an enzyme. Improved and/or altered enzymes can be identified through the development and implementation of sensitive high-throughput screening assays that allow the automated screening of many enzyme variants (e.g., >10⁴). Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme with optimized properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened.

Numerous directed evolution technologies have been developed (for reviews, see Hibbert, E. G., F. Baganz, H. C. Hailes, J. M. Ward, G. J. Lye, J. M. Woodley, and P. A. Dalby, 2005, Directed evolution of biocatalytic processes. Biomol. Eng 22:11-19; Huisman, G. W. and J. J. Lalonde, 2007, Enzyme evolution for chemical process applications, p. 717-742. In R. N. Patel (ed.), Biocatalysis in the pharmaceutical and biotechnology industries. CRC Press; Otten, L. G. and W. J. Quax. 2005. Directed evolution: selecting today's biocatalysts. Biomol. Eng 22:1-9; and Sen, S., D. Venkata, V, and B. Mandal, 2007, Developments in directed evolution for improving enzyme functions. Appl Biochem. Biotechnol 143:212-223.) to be effective at creating diverse variant libraries and these methods have been successfully applied to the improvement of a wide range of properties across many enzyme classes.

Enzyme characteristics that have been improved and/or altered by directed evolution technologies include, for example, selectivity/specificity—for conversion of non-natural substrates; temperature stability—for robust high temperature processing; pH stability—for bioprocessing under lower or higher pH conditions; substrate or product tolerance—so that high product titers can be achieved; binding (K_(m))—broadens substrate binding to include non-natural substrates; inhibition (K_(i))—to remove inhibition by products, substrates, or key intermediates; activity (kcat)—increases enzymatic reaction rates to achieve desired flux; expression levels—increases protein yields and overall pathway flux; oxygen stability—for operation of air sensitive enzymes under aerobic conditions; and anaerobic activity—for operation of an aerobic enzyme in the absence of oxygen.

The following exemplary methods have been developed for the mutagenesis and diversification of genes to target desired properties of specific enzymes. Any of these can be used to alter/optimize activity of a decarboxylase enzyme.

EpPCR (Pritchard et al. J Theor. Biol 234:497-509 (2005)) introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions by the addition of Mn²⁺ ions, by biasing dNTP concentrations, or by other conditional variations. The five step cloning process to confine the mutagenesis to the target gene of interest involves: 1) error-prone PCR amplification of the gene of interest; 2) restriction enzyme digestion; 3) gel purification of the desired DNA fragment; 4) ligation into a vector; 5) transformation of the gene variants into a suitable host and screening of the library for improved performance. This method can generate multiple mutations in a single gene simultaneously, which can be useful. A high number of mutants can be generated by EpPCR, so a high-throughput screening assay or a selection method (especially using robotics) is useful to identify those with desirable characteristics.

Error-prone Rolling Circle Amplification (epRCA) (Fujii et al., Nuc. Acids Res. 32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497 (2006)) has many of the same elements as epPCR except a whole circular plasmid is used as the template and random 6-mers with exonuclease resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re-circularized at tandem repeats. Adjusting the Mn²⁺ concentration can vary the mutation rate somewhat. This technique uses a simple error-prone, single-step method to create a full copy of the plasmid with 3-4 mutations/kbp. No restriction enzyme digestion or specific primers are required. Additionally, this method is typically available as a kit.

DNA or Family Shuffling (Stemmer, W. P., Proc Natl Acad Sci US.A 91:10747-10751 (1994); and Stemmer, W. P., Nature 370:389-391 (1994)) typically involves digestion of 2 or more variant genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes. Fragments prime each other and recombination occurs when one copy primes another copy (template switch). This method can be used with >1 kbp DNA sequences. In addition to mutational recombinants created by fragment reassembly, this method introduces point mutations in the extension steps at a rate similar to error-prone PCR. The method can be used to remove deleterious random neutral mutations that might confer antigenicity.

Staggered Extension (StEP) (Zhao et al., Nat. Biotechnol 16:258-261 (1998)) entails template priming followed by repeated cycles of 2 step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec). Growing fragments anneal to different templates and extend further, which is repeated until full-length sequences are made. Template switching means most resulting fragments have multiple parents. Combinations of low-fidelity polymerases (Taq and Mutazyme) reduce error-prone biases because of opposite mutational spectra.

In Random Priming Recombination (RPR) random sequence primers are used to generate many short DNA fragments complementary to different segments of the template. (Shao et al., Nucleic Acids Res 26:681-683 (1998)) Base misincorporation and mispriming via epPCR give point mutations. Short DNA fragments prime one another based on homology and are recombined and reassembled into full-length by repeated thermocycling. Removal of templates prior to this step assures low parental recombinants. This method, like most others, can be performed over multiple iterations to evolve distinct properties. This technology avoids sequence bias, is independent of gene length, and requires very little parent DNA for the application.

In Heteroduplex Recombination linearized plasmid DNA is used to form heteroduplexes that are repaired by mismatch repair. (Volkov et al., Nucleic Acids Res. 27:e18 (1999); and Volkov et al., Methods Enzymol. 328:456-463 (2000)) The mismatch repair step is at least somewhat mutagenic. Heteroduplexes transform more efficiently than linear homoduplexes. This method is suitable for large genes and whole operons.

Random Chimeragenesis on Transient Templates (RACHITT) (Coco et al., Nat. Biotechnol 19:354-359 (2001)) employs Dnase I fragmentation and size fractionation of ssDNA. Homologous fragments are hybridized in the absence of polymerase to a complementary ssDNA scaffold. Any overlapping unhybridized fragment ends are trimmed down by an exonuclease. Gaps between fragments are filled in, and then ligated to give a pool of full-length diverse strands hybridized to the scaffold (that contains U to preclude amplification). The scaffold then is destroyed and is replaced by a new strand complementary to the diverse strand by PCR amplification. The method involves one strand (scaffold) that is from only one parent while the priming fragments derive from other genes; the parent scaffold is selected against. Thus, no reannealing with parental fragments occurs. Overlapping fragments are trimmed with an exonuclease. Otherwise, this is conceptually similar to DNA shuffling and StEP. Therefore, there should be no siblings, few inactives, and no unshuffled parentals. This technique has advantages in that few or no parental genes are created and many more crossovers can result relative to standard DNA shuffling.

Recombined Extension on Truncated templates (RETT) entails template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates. (Lee et al., J. Molec. Catalysis 26:119-129 (2003)) No DNA endonucleases are used. Unidirectional ssDNA is made by by DNA polymerase with random primers or serial deletion with exonuclease. Unidirectional ssDNA are only templates and not primers. Random priming and exonucleases don't introduce sequence bias as true of enzymatic cleavage of DNA shuffling/RACHITT. RETT can be easier to optimize than StEP because it uses normal PCR conditions instead of very short extensions. Recombination occurs as a component of the PCR steps—no direct shuffling. This method can also be more random than StEP due to the absence of pauses.

In Degenerate Oligonucleotide Gene Shuffling (DOGS) degenerate primers are used to control recombination between molecules; (Bergquist and Gibbs, Methods Mol. Biol 352:191-204 (2007); Bergquist et al., Biomol. Eng 22:63-72 (2005); Gibbs et al., Gene 271:13-20 (2001)) this can be used to control the tendency of other methods such as DNA shuffling to regenerate parental genes. This method can be combined with random mutagenesis (epPCR) of selected gene segments. This can be a good method to block the reformation of parental sequences. No endonucleases are needed. By adjusting input concentrations of segments made, one can bias towards a desired backbone. This method allows DNA shuffling from unrelated parents without restriction enzyme digests and allows a choice of random mutagenesis methods.

Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY)creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest. (Ostermeier et al., Proc. Natl. Acad. Sci. U.S.A. 96:3562-3567 (1999); Ostermeier et al., Nat. Biotechnol 17:1205-1209 (1999)) Truncations are introduced in opposite direction on pieces of 2 different genes. These are ligated together and the fusions are cloned. This technique does not require homology between the 2 parental genes. When ITCHY is combined with DNA shuffling, the system is called SCRATCHY (see below). A major advantage of both is no need for homology between parental genes; for example, functional fusions between an E. coli and a human gene were created via ITCHY. When ITCHY libraries are made, all possible crossovers are captured.

Thio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY) is almost the same as ITCHY except that phosphothioate dNTPs are used to generate truncations. (Lutz, S., M. Ostermeier, and S. J. Benkovic, 2001, Rapid generation of incremental truncation libraries for protein engineering using alpha-phosphothioate nucleotides. Nucleic Acids Res 29:E16.) Relative to ITCHY, THIO-ITCHY can be easier to optimize, provide more reproducibility, and adjustability.

SCRATCHY-ITCHY combined with DNA shuffling is a combination of DNA shuffling and ITCHY; therefore, allowing multiple crossovers. (Lutz et al. Proc. Natl. Acad. Sci. U.S.A. 98:11248-11253 (2001)) SCRATCHY combines the best features of ITCHY and DNA shuffling. Computational predictions can be used in optimization. SCRATCHY is more effective than DNA shuffling when sequence identity is below 80%.

In Random Drift Mutagenesis (RNDM) mutations made via epPCR followed by screening/selection for those retaining usable activity. (Bergquist. Biomol. Eng 22:63-72 (2005)) Then, these are used in DOGS to generate recombinants with fusions between multiple active mutants or between active mutants and some other desirable parent. Designed to promote isolation of neutral mutations; its purpose is to screen for retained catalytic activity whether or not this activity is higher or lower than in the original gene. RNDM is usable in high throughput assays when screening is capable of detecting activity above background. RNDM has been used as a front end to DOGS in generating diversity. The technique imposes a requirement for activity prior to shuffling or other subsequent steps; neutral drift libraries are indicated to result in higher/quicker improvements in activity from smaller libraries. Though published using epPCR, this could be applied to other large-scale mutagenesis methods.

Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis method that: 1) generates pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage; this pool is used as a template to 2) extend in the presence of “universal” bases such as inosine; 3) replication of a inosine-containing complement gives random base incorporation and, consequently, mutagenesis. (Wong et al., Biotechnol J. 3:74-82 (2008); Wong et al., Nucleic Acids Res 32:e26 (2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)) Using this technique it can be possible to generate a large library of mutants within 2-3 days using simple methods. This is very non-directed compared to mutational bias of DNA polymerases. Differences in this approach makes this technique complementary (or alternative) to epPCR.

In Synthetic Shuffling, overlapping oligonucleotides are designed to encode “all genetic diversity in targets” and allow a very high diversity for the shuffled progeny. (Ness et al., Nat. Biotechnol 20:1251-1255 (2002)) In this technique, one can design the fragments to be shuffled. This aids in increaseing the resulting diversity of the progeny. One can design sequence/codon biases to make more distantly related sequences recombine at rates approaching more closely related sequences and it doesn't require possessing the template genes physically.

Nucleotide Exchange and Excision Technology NexT exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation. (Muller et al., Nucleic Acids Res 33:e117 (2005)) The gene is reassembled using internal PCR primer extension with proofreading polymerase. The sizes for shuffling are directly controllable using varying dUPT::dTTP ratios. This is an end point reaction using simple methods for uracil incorporation and cleavage. One can use other nucleotide analogs such as 8-oxo-guanine with this method. Additionally, the technique works well with very short fragments (86 bp) and has a low error rate. Chemical cleavage of DNA means very few unshuffled clones.

In Sequence Homology-Independent Protein Recombination (SHIPREC) a linker is used to facilitate fusion between 2 distantly/unrelated genes; nuclease treatment is used to generate a range of chimeras between the two. Result is a single crossover library of these fusions. (Sieber et al., Nat. Biotechnol 19:456-460 (2001)) This produces a limited type of shuffling; mutagenesis is a separate process. This technique can create a library of chimeras with varying fractions of each of 2 unrelated parent genes. No homology is needed. SHIPREC was tested with a heme-binding domain of a bacterial CP450 fused to N-terminal regions of a mammalian CP450; this produced mammalian activity in a more soluble enzyme.

In Gene Site Saturation Mutagenesis (GSSM) the starting materials are a supercoiled dsDNA plasmid with insert and 2 primers degenerate at the desired site for mutations. (Kretz et al., Methods Enzymol. 388:3-11 (2004).) Primers carry the mutation of interest and anneal to the same sequence on opposite strands of DNA; mutation in the middle of the primer and ˜20 nucleotides of correct sequence flanking on each side. The sequence in the primer is NNN or NNK (coding) and MNN (noncoding) (N=all 4, K=G, T, M=A, C). After extension, DpnI is used to digest dam-methylated DNA to eliminate the wild-type template. This technique explores all possible amino acid substitutions at a given locus (i.e., one codon). The technique facilitates the generation of all possible replacements at one site with no nonsense codons and equal or near-equal representation of most possible alleles. It does not require prior knowledge of structure, mechanism, or domains of the target enzyme. If followed by shuffling or Gene Reassembly, this technology creates a diverse library of recombinants containing all possible combinations of single-site up-mutations. The utility of this technology combination has been demonstrated for the successful evolution of over 50 different enzymes, and also for more than one property in a given enzyme.

Combinatorial Cassette Mutagenesis (CCM) involves the use of short oligonucleotide cassettes to replace limited regions with a large number of possible amino acid sequence alterations. (Reidhaar-Olson et al., Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al., Science 241:53-57 (1988).) Simultaneous substitutions at 2 or 3 sites are possible using this technique. Additionally, the method tests a large multiplicity of possible sequence changes at a limited range of sites. It has been used to explore the information content of lambda repressor DNA-binding domain.

Combinatorial Multiple Cassette Mutagenesis (CMCM) is essentially similar to CCM except it is employed as part of a larger program: 1) Use of epPCR at high mutation rate to 2) ID hot spots and hot regions and then 3) extension by CMCM to cover a defined region of protein sequence space. (Reetz et al., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001).) As with CCM, this method can test virtually all possible alterations over a target region. If used along with methods to create random mutations and shuffled genes, it provides an excellent means of generating diverse, shuffled proteins. This approach was successful in increasing, by 51-fold, the enantioselectivity of an enzyme.

In the Mutator Strains technique conditional is mutator plasmids allow increases of 20- to 4000-X in random and natural mutation frequency during selection and to block accumulation of deleterious mutations when selection is not required. (Selifonova et al., Appl Environ Microbiol 67:3645-3649 (2001).) This technology is based on a plasmid-derived mutD5 gene, which encodes a mutant subunit of DNA polymerase III. This subunit binds to endogenous DNA polymerase III and compromises the proofreading ability of polymerase III in any of the strain that harbors the plasmid. A broad-spectrum of base substitutions and frameshift mutations occur. In order for effective use, the mutator plasmid should be removed once the desired phenotype is achieved; this is accomplished through a temperature sensitive origin of replication, which allows plasmid curing at 41° C. It should be noted that mutator strains have been explored for quite some time (e.g., see Winter et al., J. Mol. Biol. 260, 359-3680 (1996). In this technique very high spontaneous mutation rates are observed. The conditional property minimizes non-desired background mutations. This technology could be combined with adaptive evolution to enhance mutagenesis rates and more rapidly achieve desired phenotypes.

“Look-Through Mutagenesis (LTM) is a multidimensional mutagenesis method that assesses and optimizes combinatorial mutations of selected amino acids.” (Rajpal et al., Proc. Natl. Acad. Sci. U.S.A. 102:8466-8471 (2005).) Rather than saturating each site with all possible amino acid changes, a set of 9 is chosen to cover the range of amino acid R-group chemistry. Fewer changes per site allows multiple sites to be subjected to this type of mutagenesis. A >800-fold increase in binding affinity for an antibody from low nanomolar to picomolar has been achieved through this method. This is a rational approach to minimize the number of random combinations and should increase the ability to find improved traits by greatly decreasing the numbers of clones to be screened. This has been applied to antibody engineering, specifically to increase the binding affinity and/or reduce dissociation. The technique can be combined with either screens or selections.

Gene Reassembly is a DNA shuffling method that can be applied to multiple genes at one time or to creating a large library of chimeras (multiple mutations) of a single gene. (on the world-wide web at verenium.com/Pages/Technology/EnzymeTech/TechEnzyTGR.html) Typically this technology is used in combination with ultra-high-throughput screening to query the represented sequence space for desired improvements. This technique allows multiple gene recombination independent of homology. The exact number and position of cross-over events can be pre-determined using fragments designed via bioinformatic analysis. This technology leads to a very high level of diversity with virtually no parental gene reformation and a low level of inactive genes. Combined with GSSM, a large range of mutations can be tested for improved activity. The method allows “blending” and “fine tuning” of DNA shuffling, e.g. codon usage can be optimized.

In Silico Protein Design Automation PDA is an optimization algorithm that anchors the structurally defined protein backbone possessing a particular fold, and searches sequence space for amino acid substitutions that can stabilize the fold and overall protein energetics. (Hayes et al., Proc. Natl. Acad. Sci. U.S.A. 99:15926-15931 (2002).) This technology allows in silico structure-based entropy predictions in order to search for structural tolerance toward protein amino acid variations. Statistical mechanics is applied to calculate coupling interactions at each position—structural tolerance toward amino acid substitution is a measure of coupling. Ultimately, this technology is designed to yield desired modifications of protein properties while maintaining the integrity of structural characteristics. The method computationally assesses and allows filtering of a very large number of possible sequence variants (10⁵⁰). Choice of sequence variants to test is related to predictions based on most favorable thermodynamics and ostensibly only stability or properties that are linked to stability can be effectively addressed with this technology. The method has been successfully used in some therapeutic proteins, especially in engineering immunoglobulins. In silico predictions avoid testing extraordinarily large numbers of potential variants. Predictions based on existing three-dimensional structures are more likely to succeed than predictions based on hypothetical structures. This technology can readily predict and allow targeted screening of multiple simultaneous mutations, something not possible with purely experimental technologies due to exponential increases in numbers.

Iterative Saturation Mutagenesis (ISM)involves 1) Use knowledge of structure/function to choose a likely site for enzyme improvement. 2) Saturation mutagenesis at chosen site using Stratagene QuikChange (or other suitable means). 3) Screen/select for desired properties. 4) With improved clone(s), start over at another site and continue repeating. (Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et al., Angew. Chem. Int. Ed Engl. 45:7745-7751 (2006).) This is a proven methodology assures all possible replacements at a given position are made for screening/selection.

Any of the aforementioned methods for mutagenesis can be used alone or in any combination. Additionally, any one or combination of the directed evolution methods can be used in conjunction with adaptive evolution techniques.

To generate better producers, metabolic modeling can be utilized to optimize growth conditions. Modeling can also be used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of MEK or 2-butanol.

One computational method for identifying and designing metabolic alterations favoring biosynthesis of a desired product is the OptKnock computational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)). OptKnock is a metabolic modeling and simulation program that suggests gene deletion or disruption strategies that result in genetically stable microorganisms which overproduce the target product. Specifically, the framework examines the complete metabolic and/or biochemical network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become an obligatory byproduct of cell growth. By coupling biochemical production with cell growth through strategically placed gene deletions or other functional gene disruption, the growth selection pressures imposed on the engineered strains after long periods of time in a bioreactor lead to improvements in performance as a result of the compulsory growth-coupled biochemical production. Lastly, when gene deletions are constructed there is a negligible possibility of the designed strains reverting to their wild-type states because the genes selected by OptKnock are to be completely removed from the genome. Therefore, this computational methodology can be used to either identify alternative pathways that lead to biosynthesis of a desired product or used in connection with the non-naturally occurring microbial organisms for further optimization of biosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism. The OptKnock program relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) models. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data. OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or deletions. OptKnock computational framework allows the construction of model formulations that enable an effective query of the performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems. The metabolic modeling and simulation methods referred to herein as OptKnock are described in, for example, U.S. publication 2002/0168654, filed Jan. 10, 2002, in International Patent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. publication 2009/0047719, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolic alterations favoring biosynthetic production of a product is a metabolic modeling and simulation system termed SimPheny®. This computational method and system is described in, for example, U.S. publication 2003/0233218, filed Jun. 14, 2002, and in International Patent Application No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system. This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realities because biological systems are flexible and can reach the same result in many different ways. Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a reduction in the size of the solution space, thereby enhancing the precision with which physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in the art will be able to apply various computational frameworks for metabolic modeling and simulation to design and implement biosynthesis of a desired compound in host microbial organisms. Such metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny® and OptKnock. For illustration of the invention, some methods are described herein with reference to the OptKnock computation framework for modeling and simulation. Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation computational frameworks and methods well known in the art.

The methods described above will provide one set of metabolic reactions to disrupt. Elimination of each reaction within the set or metabolic modification can result in a desired product as an obligatory product during the growth phase of the organism. Because the reactions are known, a solution to the bilevel OptKnock problem also will provide the associated gene or genes encoding one or more enzymes that catalyze each reaction within the set of reactions. Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction database having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in order to achieve production of a desired product are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set. One particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene. However, in some instances, it can be beneficial to disrupt the reaction by other genetic aberrations including, for example, mutation, deletion of regulatory regions such as promoters or cis binding sites for regulatory factors, or by truncation of the coding sequence at any of a number of locations. These latter aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid assessments of the coupling of a product are desired or when genetic reversion is less likely to occur.

To identify additional productive solutions to the above described bilevel OptKnock problem which lead to further sets of reactions to disrupt or metabolic modifications that can result in the biosynthesis, including growth-coupled biosynthesis of a desired product, an optimization method, termed integer cuts, can be implemented. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that obligatorily couples product biosynthesis to growth. For example, if a previously identified growth-coupled metabolic modification specifies reactions 1, 2, and 3 for disruption, then the following constraint prevents the same reactions from being simultaneously considered in subsequent solutions. The integer cut method is well known in the art and can be found described in, for example, Burgard et al., Biotechnol. Prog. 17:791-797 (2001). As with all methods described herein with reference to their use in combination with the OptKnock computational framework for metabolic modeling and simulation, the integer cut method of reducing redundancy in iterative computational analysis also can be applied with other computational frameworks well known in the art including, for example, SimPheny®.

The methods exemplified herein allow the construction of cells and organisms that biosynthetically produce a desired product, including the obligatory coupling of production of a target biochemical product to growth of the cell or organism engineered to harbor the identified genetic alterations. Therefore, the computational methods described herein allow the identification and implementation of metabolic modifications that are identified by an in silico method selected from OptKnock or SimPheny®. The set of metabolic modifications can include, for example, addition of one or more biosynthetic pathway enzymes and/or functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on the premise that mutant microbial networks can be evolved towards their computationally predicted maximum-growth phenotypes when subjected to long periods of growth selection. In other words, the approach leverages an organism's ability to self-optimize under selective pressures. The OptKnock framework allows for the exhaustive enumeration of gene deletion combinations that force a coupling between biochemical production and cell growth based on network stoichiometry. The identification of optimal gene/reaction knockouts requires the solution of a bilevel optimization problem that chooses the set of active reactions such that an optimal growth solution for the resulting network overproduces the biochemical of interest (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employed to identify essential genes for metabolic pathways as exemplified previously and described in, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No. 7,127,379. As disclosed herein, the OptKnock mathematical framework can be applied to pinpoint gene deletions leading to the growth-coupled production of a desired product. Further, the solution of the bilevel OptKnock problem provides only one set of deletions. To enumerate all meaningful solutions, that is, all sets of knockouts leading to growth-coupled production formation, an optimization technique, termed integer cuts, can be implemented. This entails iteratively solving the OptKnock problem with the incorporation of an additional constraint referred to as an integer cut at each iteration, as discussed above.

It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also included within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.

Example I MEK Synthesis Using Acetoacetyl-CoA as the Intermediate

This Example describes the generation of a microbial organism capable of producing MEK using acetoacetyl-CoA as the precursor (Steps A, D and E in FIG. 1). Genes for the production of MEK are inserted into Escherichia coli.

Escherichia coli is used as a target organism to engineer the pathway in FIG. 1. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing MEK. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce MEK, nucleic acids encoding the enzymes utilized in the disclosed pathway (Steps A, D and E) as described previously, are expressed in E. coli using well known molecular biology methods (Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999); Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001)). Note that E. coli has a native thiolase encoded by atoB (Accession number: NP_(—)416728.1) that condenses two molecules of acetyl-CoA to form acetoacetyl-CoA. Further, adhE2 (AAK09379.1), hmd (ABC88407.1), and YML131 W (AAS56318.1), genes encoding acetoacetyl-CoA dehydrogenase (bifunctional), 3-oxobutanol dehydratase and MEK oxidoreductase activities respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. The plasmid is transformed into E. coli strain MG1655 to express the proteins and enzymes required for MEK synthesis via acetoacetyl-CoA. For increased availability of acetoacetyl-CoA, the atoB gene can be expressed on the same vector or another compatible plasmid and transformed in to E. coli as well.

The resulting genetically engineered organism is cultured in glucose containing medium following methods well known in the art (see, for example, (Ausubel et al., supra; Sambrook et al., supra)). The expression of the pathway genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including, for example, Northern blots, PCR amplification of mRNA, immunoblotting. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individually activities. The ability of the engineered E. coli strain to produce MEK is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional MEK synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of MEK. One modeling method is the bi-level optimization approach, OptKnock (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of MEK. Adaptive evolution also can be used to generate better producers of, for example, the acetoacetyl-CoA intermediate or the MEK product. Adaptive evolution is performed to improve both growth and production characteristics (Alper et al., Science 314:1565-1568 (2006); Fong and Palsson, Nat Genet. 36:1056-1058 (2004)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the MEK producer to further increase production.

For large-scale production of MEK, the recombinant organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing culture vessel (e.g., flasks can be sealed with a septum and crimp-cap). Microaerobic conditions also can be utilized by providing a small hole for limited aeration. The pH of the medium is maintained at a pH of 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu) with an HPX-087 column (BioRad), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-77 (2005)).

Example II MEK Synthesis Using Alanine as the Intermediate

This example describes the generation of a microbial organism capable of producing MEK using the alanine pathway in FIG. 2 via Steps A, B, C, D, E and J. Genes for the production of MEK are inserted into Escherichia coli.

Escherichia coli is used as a target organism to engineer a MEK-producing pathway as shown in FIG. 2. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing MEK. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce MEK, nucleic acids encoding the enzymes utilized in the alanine pathway, as described previously, are expressed in E. coli using well known molecular biology methods (see, for example, (Ausubel et al., supra; Sambrook et al., supra)). In particular, the ortA (YP_(—)001086914.1), ortB (YP_(—)001086915.1), dat (P19938), and pdc (P06672) genes encoding the AKP thiolase, AKP aminotransferase and 2,4-dioxopentanoate decarboxylase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the yqhD (NP_(—)417484.1), hmd (ABC88407.1), YML131W (AAS56318.1) and GDH2 (NP_(—)010066.1) genes encoding 3-oxobutyraldehdye reductase, 3-oxobutanol dehydratase, MEK oxidoreductase and glutamate dehydrogenase activities respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. The two sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for MEK synthesis via the alanine pathway. Note that E. coli possesses the ability to form D-alanine. However, D-alanine levels can potentially be increased by expressing alr (NP_(—)418477) or dadX (AAC74274), which encode alanine racemases, on either of the above vectors or on a third compatible plasmid and transforming into E. coli.

The resulting genetically engineered organism is cultured in glucose containing medium following methods well known in the art (see, for example, (Ausubel et al., supra; Sambrook et al., supra)). The expression of alanine pathway genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individually activities. The ability of the engineered E. coli strain to produce MEK is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional MEK synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of MEK. One modeling method is the bi-level optimization approach, OptKnock (Burgard et al., Biotechnol. Bioeng. 84:847-657 (2003)), which is applied to select gene knockouts that collectively result in better production of MEK. Adaptive evolution also can be used to generate better producers of, for example, alanine or 2-amino-4-oxopentanoate intermediates or the MEK product. Adaptive evolution is performed to improve both growth and production characteristics (Alper et al., Science 314:1565-1568 (2006); Fong and Palsson, Nat Genet. 36:1056-1058 (2004)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the MEK producer to further increase production.

For large-scale production of MEK, the above alanine pathway-containing organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing culture vessel (e.g., flasks can be sealed with a septum and crimp-cap). Microaerobic conditions also can be utilized by providing a small hole for limited aeration. The pH of the medium is maintained at a pH of 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu) with an HPX-087 column (BioRad), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids, Lin et al., Biotechnol. Bioeng., 90:775-779 (2005).

Example III 2-Butanol Synthesis Using Acetoacetyl-CoA as the Intermediate

This Example describes the generation of a microbial organism capable of producing 2-butanol using acetoacetyl-CoA as the precursor (Steps A, D and E in FIG. 1). Genes for the production of 2-butanol are inserted into Escherichia coli.

Escherichia coli is used as a target organism to engineer the pathway in FIG. 1. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing 2-butanol. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce 2-butanol, nucleic acids encoding the enzymes utilized in the disclosed pathway (Steps A, D and E) as described previously, are expressed in E. coli using well known molecular biology methods (Ausubel et al., supra; Sambrook et al., supra). Note that E. coli has a native thiolase encoded by atoB (Accession number: NP_(—)416728.1) that condenses two molecules of acetyl-CoA to form acetoacetyl-CoA. Further, adhE2 (AAK09379.1), hmd (ABC88407.1), YML131W (AAS56318.1) and sadh (CAD36475) genes encoding acetoacetyl-CoA dehydrogenase (bifunctional), 3-oxobutanol dehydratase MEK oxidoreductase and MEK reductase enzymes required for 2-butanol synthesis via acetoacetyl-CoA. For increased availability of acetoacetyl-CoA, the atoB gene can be expressed on the same vector or another compatible plasmid and transformed in to E. coli as well.

The resulting genetically engineered organism is cultured in glucose containing medium following methods well known in the art (see, for example, (Ausubel et al., supra; Sambrook et al., supra). The expression of the pathway genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including, for example, Northern blots, PCR amplification of mRNA, immunoblotting. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individually activities. The ability of the engineered E. coli strain to produce 2-butanol is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional 2-butanol synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of 2-butanol. One modeling method is the bi-level optimization approach, OptKnock ((Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of MEK. Adaptive evolution also can be used to generate better producers of, for example, the acetoacetyl-CoA intermediate or the 2-butanol product. Adaptive evolution is performed to improve both growth and production characteristics (Alper et al., Science 314:1565-1568 (2006); Fong and Palsson, Nat Genet. 36:1056-1058 (2004)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the 2-butanol producer to further increase production.

For large-scale production of 2-butanol, the recombinant organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing culture vessel (e.g., flasks can be sealed with a septum and crimp-cap). Microaerobic conditions also can be utilized by providing a small hole for limited aeration. The pH of the medium is maintained at a pH of 7 by addition of an acid, such as H2504. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu) with an HPX-087 column (BioRad), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)).

Example IV 2-Butanol Synthesis Using Alanine as the Intermediate

This example describes the generation of a microbial organism capable of producing 2-butanol using the alanine pathway in FIG. 2 via Steps A, B, C, D, E and J. Genes for the production of 2-butanol are inserted into Escherichia coli.

Escherichia coli is used as a target organism to engineer a 2-butanol -producing pathway as shown in FIG. 2. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing 2-butanol. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce 2-butanol, nucleic acids encoding the enzymes utilized in the alanine pathway, as described previously, are expressed in E. coli using well known molecular biology methods (see, for example, (Ausubel et al., supra; Sambrook et al., supra). In particular, the ortA (YP_(—)001086914.1), ortB (YP_(—)001086915.1), dat (P19938), and pdc (P06672) genes encoding the AKP thiolase, AKP aminotransferase and 2,4-dioxopentanoate decarboxylase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the yqhD (NP_(—)417484.1), hmd (ABC88407.1), YML131W(AAS56318.1), sadh (CAD36475) and GDH2 (NP_(—)010066.1) genes encoding 3-oxobutyraldehdye reductase, 3-oxobutanol dehydratase, MEK oxidoreductase MEK reductase and glutamate dehydrogenase, respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. The two sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for MEK synthesis via the alanine pathway. Note that E. coli possesses the ability to form D-alanine. However, D-alanine levels can potentially be increased by expressing alr (NP_(—)418477) or dadX (AAC74274), which encode alanine racemases, on either of the above vectors or on a third compatible plasmid and transforming into E. coli.

The resulting genetically engineered organism is cultured in glucose containing medium following methods well known in the art (see, for example,(Ausubel et al., supra; Sambrook et al., supra). The expression of alanine pathway genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individually activities. The ability of the engineered E. coli strain to produce 2-butanol is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional 2-butanol synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of 2-butanol. One modeling method is the bi-level optimization approach, OptKnock (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of MEK. Adaptive evolution also can be used to generate better producers of, for example, alanine or 2-amino-4-oxopentanoate intermediates or the 2-butanol product. Adaptive evolution is performed to improve both growth and production characteristics (Alper et al., Science 314:1565-1568 (2006); Fong and Palsson, Nat Genet. 36:1056-1058 (2004)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the 2-butanol producer to further increase production.

For large-scale production of 2-butanol, the above alanine pathway-containing organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing culture vessel (e.g., flasks can be sealed with a septum and crimp-cap). Microaerobic conditions also can be utilized by providing a small hole for limited aeration. The pH of the medium is maintained at a pH of 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu) with an HPX-087 column (BioRad), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids, Lin et al., Biotechnol. Bioeng., 775-779 (2005).

Throughout this application various publications have been referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.

Although the invention has been described with reference to the disclosed embodiments, those skilled in the art will readily appreciate that the specific examples and studies detailed above are only illustrative of the invention. It should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. 

1. A non-naturally occurring microbial organism, comprising a microbial organism having a MEK pathway comprising at least one exogenous nucleic acid encoding a MEK pathway enzyme expressed in a sufficient amount to produce MEK, said MEK pathway comprising an enzyme selected from the group consisting of an acetoacetyl-CoA dehydrogenase (bifunctional), an acetoacetyl-CoA aldehyde dehydrogenase, a 3-oxobutyraldehyde reductase, a 3-oxobutanol dehydratase, an MEK oxidoreductase, a 3-oxobutyraldehyde aminotransferase, a 4-aminobutan-2-one deaminase, a 2-amino-4-ketopentanoate (AKP) thiolase, an AKP aminotransferase, a 2,4-dioxopentanoate decarboxylase, an AKP deaminase, an acetylacrylate decarboxylase, an AKP decarboxylase, a glutamate dehydrogenase, a 3-oxobutyraldehyde oxidoreductase (aminating) and an AKP oxidoreductase (aminating), wherein said MEK pathway comprises a set of MEK pathway enzymes, said set of MEK pathway enzymes selected from the group consisting of: (1) acetoacetyl-CoA dehydrogenase (bifunctional), (2) 3-oxobutanol dehydratase, and (3) MEK oxidoreductase (1) acetoacetyl-CoA aldehyde dehydrogenase, (2) 3-oxobutyraldehyde reductase, (3) 3-oxobutanol dehydratase, and (4) MEK oxidoreductase (1) acetoacetyl-CoA aldehyde dehydrogenase, (2) 3-oxobutyraldehyde aminotransferase and/or 3-oxobutyraldehyde oxidoreductase (aminating), (3) 4-aminobutan-2-one deaminase, and (4) MEK oxidoreductase (1) AKP thiolase, (2) AKP aminotransferase and/or AKP oxidoreductase (aminating), (3) 2,4-dioxopentanoate decarboxylase, (4) 3-oxobutyraldehyde reductase, (5) 3-oxobutanol dehydratase, (6) MEK oxidoreductase, and (7) glutamate dehydrogenase (1) AKP thiolase, (2) AKP deaminase, (3) acetylacrylate decarboxylase, (4) MEK oxidoreductase and (5) glutamate dehydrogenase (1) AKP thiolase, (2) AKP decarboxylase, (3) 4-aminobutan-2-one deaminase, (4) MEK oxidoreductase and (5) glutamate dehydrogenase.
 2. (canceled)
 3. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises two exogenous nucleic acids each encoding a MEK pathway enzyme.
 4. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises three exogenous nucleic acids each encoding a MEK pathway enzyme.
 5. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises four exogenous nucleic acids each encoding a MEK pathway enzyme.
 6. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises five exogenous nucleic acids each encoding a MEK pathway enzyme.
 7. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises six exogenous nucleic acids each encoding a MEK pathway enzyme.
 8. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises seven exogenous nucleic acids each encoding a MEK pathway enzyme. 9.-24. (canceled)
 25. The non-naturally occurring microbial organism of claims 1, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.
 26. The non-naturally occurring microbial organism of claim 1, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
 27. A non-naturally occurring microbial organism, comprising a microbial organism having a 2-butanol pathway comprising at least one exogenous nucleic acid encoding a 2-butanol pathway enzyme expressed in a sufficient amount to produce 2-butanol, said 2-butanol pathway comprising an enzyme selected from the group consisting of an acetoacetyl-CoA dehydrogenase (bifunctional), an acetoacetyl-CoA aldehyde dehydrogenase, a 3-oxobutyraldehyde reductase, a 3-oxobutanol dehydratase, an MEK oxidoreductase, an MEK reductase, an 3-oxobutyraldehyde aminotransferase, a 4-aminobutan-2-one deaminase, a 2-amino-4-ketopentanoate (AKP) thiolase, an AKP aminotransferase, a 2,4-dioxopentanoate decarboxylase, an AKP deaminase, an acetylacrylate decarboxylase, an AKP decarboxylase, a glutamate dehydrogenase, a 3-oxobutyraldehyde oxidoreductase (aminating) and an AKP oxidoreductase (aminating), wherein said 2-butanol pathway comprises a set of 2-butanol pathway enzymes, said set of 2-butanol pathway enzymes selected from the group consisting of: (1) acetoacetyl-CoA dehydrogenase (bifunctional), (2) 3-oxobutanol dehydratase, (3) MEK oxidoreductase, and (4) MEK reductase (1) acetoacetyl-CoA aldehyde dehydrogenase, (2) 3-oxobutyraldehyde reductase, (3) 3-oxobutanol dehydratase, (4) MEK oxidoreductase, and (5) MEK reductase (1) acetoacetyl-CoA aldehyde dehydrogenase, (2) 3-oxobutyraldehyde transaminase and/or 3-oxobutyraldehyde oxidoreductase (aminating), (3) 4-aminobutan-2-one deaminase, (4) MEK oxidoreductase, and (5) MEK reductase (1) AKP thiolase, (2) AKP aminotransferase and/or AKP oxidoreductase (aminating), (3) 2,4-dioxopentanoate decarboxylase, (4) 3-oxobutyraldehyde reductase, (5) 3-oxobutanol dehydratase, (6) MEK oxidoreductase, (7) MEK reductase and (8) glutamate dehydrogenase. (1) AKP thiolase, (2) AKP deaminase, (3) acetylacrylate decarboxylase, (4) MEK oxidoreductase, (5) MEK reductase and (6) glutamate dehydrogenase (1) AKP thiolase, (2) AKP decarboxylase, (3) 4-aminobutan-2-one deaminase, (4) MEK oxidoreductase, (5) MEK reductase and (6) glutamate dehydrogenase.
 28. (canceled)
 29. The non-naturally occurring microbial organism of claim 27, wherein said microbial organism comprises two exogenous nucleic acids each encoding a 2-butanol pathway enzyme.
 30. The non-naturally occurring microbial organism of claim 27, wherein said microbial organism comprises three exogenous nucleic acids each encoding a 2-butanol pathway enzyme.
 31. The non-naturally occurring microbial organism of claim 27, wherein said microbial organism comprises four exogenous nucleic acids each encoding a 2-butanol pathway enzyme.
 32. The non-naturally occurring microbial organism of claim 27, wherein said microbial organism comprises five exogenous nucleic acids each encoding a 2-butanol pathway enzyme.
 33. The non-naturally occurring microbial organism of claim 27, wherein said microbial organism comprises six exogenous nucleic acids each encoding a 2-butanol pathway enzyme.
 34. The non-naturally occurring microbial organism of claim 27, wherein said microbial organism comprises seven exogenous nucleic acids each encoding a 2-butanol pathway enzyme.
 35. The non-naturally occurring microbial organism of claim 27, wherein said microbial organism comprises eight exogenous nucleic acids each encoding a 2-butanol pathway enzyme. 36.-52. (canceled)
 53. The non-naturally occurring microbial organism of claim 27, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.
 54. The non-naturally occurring microbial organism of claim 27, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
 55. A method for producing MEK comprising culturing a non-naturally occurring microbial organism having a MEK pathway, said pathway comprising at least one exogenous nucleic acid encoding a MEK pathway enzyme expressed in a sufficient amount to produce MEK under conditions and for a sufficient period of time to produce MEK said MEK pathway comprising an enzyme selected from the group consisting of an acetoacetyl-CoA dehydrogenase (bifunctional), an acetoacetyl-CoA aldehyde dehydrogenase, a 3-oxobutyraldehyde reductase, a 3-oxobutanol dehydratase, an MEK oxidoreductase, a 3-oxobutyraldehyde aminotransferase, a 4-aminobutan-2-one deaminase, a 2-amino-4-ketopentanoate (AKP) thiolase, an AKP aminotransferase, a 2,4-dioxopentanoate decarboxylase, an AKP deaminase, an acetylacrylate decarboxylase, an AKP decarboxylase, a glutamate dehydrogenase, a 3-oxobutyraldehyde oxidoreductase (aminating) and an AKP oxidoreductase (aminating), wherein said MEK pathway comprises a set of MEK pathway enzymes, said set of MEK pathway enzymes selected from the group consisting of: (1) acetoacetyl-CoA dehydrogenase (bifunctional), (2) 3-oxobutanol dehydratase, and (3) MEK oxidoreductase (1) acetoacetyl-CoA aldehyde dehydrogenase, (2) 3-oxobutyraldehyde reductase, (3) 3-oxobutanol dehydratase, and (4) MEK oxidoreductase (1) acetoacetyl-CoA aldehyde dehydrogenase, (2) 3-oxobutyraldehyde aminotransferase and/or 3-oxobutyraldehyde oxidoreductase (aminating), (3) 4-aminobutan-2-one deaminase, and (4) MEK oxidoreductase (1) AKP thiolase, (2) AKP aminotransferase and/or AKP oxidoreductase (aminating), (3) 2,4-dioxopentanoate decarboxylase, (4) 3-oxobutyraldehyde reductase, (5) 3-oxobutanol dehydratase, (6) MEK oxidoreductase, and (7) glutamate dehydrogenase (1) AKP thiolase, (2) AKP deaminase, (3) acetylacrylate decarboxylase, (4) MEK oxidoreductase and (5) glutamate dehydrogenase (1) AKP thiolase, (2) AKP decarboxylase, (3) 4-aminobutan-2-one deaminase, (4) MEK oxidoreductase and (5) glutamate dehydrogenase.
 56. (canceled)
 57. The method of claim 55, wherein said microbial organism comprises two exogenous nucleic acids each encoding a MEK pathway enzyme.
 58. The method of claim 55, wherein said microbial organism comprises three exogenous nucleic acids each encoding a MEK pathway enzyme.
 59. The method of claim 55, wherein said microbial organism comprises four exogenous nucleic acids each encoding a MEK pathway enzyme.
 60. The method of claim 55, wherein said microbial organism comprises five exogenous nucleic acids each encoding a MEK pathway enzyme.
 61. The method of claim 55, wherein said microbial organism comprises six exogenous nucleic acids each encoding a MEK pathway enzyme.
 62. The method of claim 55, wherein said microbial organism comprises seven exogenous nucleic acids each encoding a MEK pathway enzyme. 63.-78. (canceled)
 79. The method of claim 55, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.
 80. The method of claim 55, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
 81. A method for producing 2-butanol comprising culturing a non-naturally occurring microbial organism having a 2-butanol pathway, said pathway comprising at least one exogenous nucleic acid encoding a 2-butanol pathway enzyme expressed in a sufficient amount to produce 2-butanol under conditions and for a sufficient period of time to produce 2-butanol said 2-butanol pathway comprising an enzyme selected from the group consisting of an acetoacetyl-CoA dehydrogenase (bifunctional), an acetoacetyl-CoA aldehyde dehydrogenase, a 3-oxobutyraldehyde reductase, a 3-oxobutanol dehydratase, an MEK oxidoreductase, an MEK reductase, an 3-oxobutyraldehyde aminotransferase, a 4-aminobutan-2-one deaminase, a 2-amino-4-ketopentanoate (AKP) thiolase, an AKP aminotransferase, a 2,4-dioxopentanoate decarboxylase, an AKP deaminase, an acetylacrylate decarboxylase, an AKP decarboxylase, a glutamate dehydrogenase, a 3-oxobutyraldehyde oxidoreductase (aminating) and an AKP oxidoreductase (aminating), wherein said 2-butanol pathway comprises a set of 2-butanol pathway enzymes, said set of 2-butanol pathway enzymes selected from the group consisting of: (1) acetoacetyl-CoA dehydrogenase (bifunctional), (2) 3-oxobutanol dehydratase, (3) MEK oxidoreductase, and (4) MEK reductase (1) acetoacetyl-CoA aldehyde dehydrogenase, (2) 3-oxobutyraldehyde reductase, (3) 3-oxobutanol dehydratase, (4) MEK oxidoreductase, and (5) MEK reductase (1) acetoacetyl-CoA aldehyde dehydrogenase, (2) 3-oxobutyraldehyde transaminase and/or 3-oxobutyraldehyde oxidoreductase (aminating), (3) 4-aminobutan-2-one deaminase, (4) MEK oxidoreductase, and (5) MEK reductase (1) AKP thiolase, (2) AKP aminotransferase and/or AKP oxidoreductase (aminating), (3) 2,4-dioxopentanoate decarboxylase, (4) 3-oxobutyraldehyde reductase, (5) 3-oxobutanol dehydratase, (6) MEK oxidoreductase, (7) MEK reductase and (8) glutamate dehydrogenase. (1) AKP thiolase, (2) AKP deaminase, (3) acetylacrylate decarboxylase, (4) MEK oxidoreductase, (5) MEK reductase and (6) glutamate dehydrogenase (1) AKP thiolase, (2) AKP decarboxylase, (3) 4-aminobutan-2-one deaminase, (4) MEK oxidoreductase, (5) MEK reductase and (6) glutamate dehydrogenase.
 82. (canceled)
 83. The method of claim 81, wherein said microbial organism comprises two exogenous nucleic acids each encoding a 2-butanol pathway enzyme.
 84. The method of claim 81, wherein said microbial organism comprises three exogenous nucleic acids each encoding a 2-butanol pathway enzyme.
 85. The method of claim 81, wherein said microbial organism comprises four exogenous nucleic acids each encoding a 2-butanol pathway enzyme.
 86. The method of claim 81, wherein said microbial organism comprises five exogenous nucleic acids each encoding a 2-butanol pathway enzyme.
 87. The method of claim 81, wherein said microbial organism comprises six exogenous nucleic acids each encoding a 2-butanol pathway enzyme.
 88. The method of claim 81, wherein said microbial organism comprises seven exogenous nucleic acids each encoding a 2-butanol pathway enzyme.
 89. The method of claim 81, wherein said microbial organism comprises eight exogenous nucleic acids each encoding a 2-butanol pathway enzyme. 90.-106. (canceled)
 107. The method of claim 81, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.
 108. The method of claim 81, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 